U.S. patent application number 16/337951 was filed with the patent office on 2019-09-19 for use of ahr agonist for the preventive or curative treatment of metabolic syndrome and the associated disorders.
The applicant listed for this patent is ASSISTANCE PUBLIQUE - HOPITAUX DE PARIS, INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE, INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE, SORBONNE UNIVERSITE. Invention is credited to HENRI DUBOC, BRUNO LAMAS, PHILIPPE LANGELLA, MATHIAS LAVIE-RICHARD, MARIE-LAURE MICHEL, JANE MEA NATIVIDAD, HARRY SOKOL.
Application Number | 20190282638 16/337951 |
Document ID | / |
Family ID | 57144927 |
Filed Date | 2019-09-19 |
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United States Patent
Application |
20190282638 |
Kind Code |
A1 |
SOKOL; HARRY ; et
al. |
September 19, 2019 |
USE OF AHR AGONIST FOR THE PREVENTIVE OR CURATIVE TREATMENT OF
METABOLIC SYNDROME AND THE ASSOCIATED DISORDERS
Abstract
The present invention relates to the preventive or curative
treatment of metabolic syndrome and the associated disorders with
AhR agonist or microorganism producing AhR agonist.
Inventors: |
SOKOL; HARRY; (PARIS,
FR) ; NATIVIDAD; JANE MEA; (PARIS, FR) ;
LAMAS; BRUNO; (MASSY, FR) ; DUBOC; HENRI;
(VERSAILLES, FR) ; LAVIE-RICHARD; MATHIAS; (SAINT
CYR L'ECOLE, FR) ; MICHEL; MARIE-LAURE; (VERSAILLES,
FR) ; LANGELLA; PHILIPPE; (VELIZY, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE
SORBONNE UNIVERSITE
ASSISTANCE PUBLIQUE - HOPITAUX DE PARIS
INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE |
PARIS 7
PARIS
PARIS
PARIS |
|
FR
FR
FR
FR |
|
|
Family ID: |
57144927 |
Appl. No.: |
16/337951 |
Filed: |
July 11, 2017 |
PCT Filed: |
July 11, 2017 |
PCT NO: |
PCT/EP2017/067438 |
371 Date: |
March 29, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/747 20130101;
A61K 31/167 20130101; A61K 31/42 20130101; A61P 3/10 20180101; A61K
31/138 20130101; A61K 31/40 20130101; A61K 31/44 20130101; A61K
31/404 20130101; A61K 31/4045 20130101; A61K 35/745 20130101; A61P
3/04 20180101; A61K 9/0053 20130101; A61K 35/744 20130101; A61K
36/258 20130101; A61P 9/00 20180101; Y02A 50/30 20180101; Y02A
50/473 20180101; A61P 1/16 20180101 |
International
Class: |
A61K 35/747 20060101
A61K035/747; A61K 31/138 20060101 A61K031/138; A61K 31/44 20060101
A61K031/44; A61K 31/167 20060101 A61K031/167; A61K 31/40 20060101
A61K031/40; A61K 31/42 20060101 A61K031/42; A61K 36/258 20060101
A61K036/258; A61P 3/10 20060101 A61P003/10; A61P 3/04 20060101
A61P003/04; A61P 1/16 20060101 A61P001/16 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 2016 |
EP |
16306300.1 |
Claims
1-13. (canceled)
14. A method for the preventive or curative treatment of metabolic
syndrome and the associated disorders in a subject comprising
administering a bacterial probiotic that produces an aryl
hydrocarbon receptor (AhR) agonist or an aryl hydrocarbon receptor
(AhR) agonist to a subject.
15. The method according to claim 14, wherein the associated
disorders are selected from the group consisting of cardiovascular
disease, in particular coronary heart disease, especially heart
attack and stroke, insulin resistance, glucose intolerance, type 2
diabetes, fatty liver disease, and lipodystrophy.
16. The method according to claim 14, wherein the subject exhibits
decreased AhR activity or decreased AhR activity of gut
microbiota.
17. The method according to claim 14, wherein the AhR agonist is
selected from the group consisting of indole derivatives,
tryptophan catabolites of the microbiota, kynurenine, kynurenic
acid, indole-3-aldehyde (IAld), tryptamine, indole 3-acetate,
3-indoxyl sulfate, 6-formylindolo(3,2-b)carbazole (FICZ),
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tryptophan derivatives,
flavonoids, biphenyls, AhR modulator (SAhRM), diindolylmethane
(DIM), methyl-substituted diindolylmethanes, dihalo- and dialkylDIM
analogs, mexiletine, polycyclic aromatic hydrocarbon (PAH),
polychlorinated biphenyl (PCB), .beta.-naphthoflavone (3NF), 5,6
benzoflavone (5,6 BZF), 3-indoxyl-sulfate
(13S),1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazol-
yl)ethanone hydrobromide (Pifithrin-.alpha. hydrobromide),
(2'Z,3'E)-6-Bromo-1-methylindirubin-3'-oxime (MeB10),
5-hydroxy-7-methoxyflavone, 7-methoxyisoflavone, 6-methylflavone,
3-hydroxy-6-methylflavone, pinocembrin (5,7-dihydroxyflavanone) and
7,8,2'-trihydroxyflavone, 1,4-dihydroxy-2-naphthoic acid (DHNA),
SU5416, CB7950998, Nimidipine, Flutamide, Atorvastatin,
Leflunomide, Ginseng and natural AhR Agonists (NAhRAs).
18. The method according to claim 14, wherein the bacterial
probiotic is a bacterium naturally producing AhR agonist or a
genetically modified bacterium producing an AhR agonist.
19. The method according to claim 18, wherein said bacterium is an
Allobaculum, Lactobacillus, Adlercreutzia, Actinobacteria, lactic
acid bacterium, Streptococcus thermophilus, Bifidobacterium,
Propionic acid bacterium, Bacteroides, Eubacterium, anaerobic
Streptococcus, Anaerostipes or Enterococcus.
20. The method according to claim 18, wherein said bacterium is
Allobaculum stercoricanis, Lactobacillus reuteri, Lactobacillus
taiwanensis, Lactobacillus johnsonii, Lactobacillus animalis,
Lactobacillus murinus, Lactobacillus salivarius, Lactobacillus
gasseri, Lactobacillus bulgaricus, Lactobacillus delbrueckii subsp.
Bulgaricus, Streptococcus thermophilus, Anaerostipes hadrus,
Anaerostipes caccae, Anaerostipes butyraticus, Ruminococcus gnavus,
Faecalibacterium prausnitzii or Escherichia coli.
21. The method according to claim 19, wherein the bacterial
probiotic is an Allobaculum.
22. The method according to claim 19, wherein the bacterial
probiotic is a Lactobacillus.
23. The method according to claim 22, wherein the bacterial
probiotic is selected from the group consisting of Lactobacillus
reuteri, Lactobacillus taiwanensis, Lactobacillus animalis,
Lactobacillus murinus, Lactobacillus salivarius, Lactobacillus
gasseri, Lactobacillus bulgaricus, and Lactobacillus delbrueckii
subsp. Bulgaricus.
24. The method according to claim 14, wherein the bacterial
probiotic is selected from the group consisting of bacterial
probiotics available under CNCM deposit numbers CNCM I-5019, CNCM
I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023 and any combination
thereof.
25. The method according to claim 18, wherein the bacterial
probiotic is Lactobacillus delbrueckii subsp. Bulgaricus or is
Lactobacillus delbrueckii subsp. Bulgaricus OLL1181.
26. The method according to claim 14, wherein the bacterial
probiotic is administered orally or rectally.
27. The method according to claim 14, wherein the AhR agonist is
administered enterally or parenterally.
28. The method according to claim 14, wherein the bacterial
probiotic is selected from the group consisting of a bacterial
probiotics deposited under CNCM deposit numbers CNCM I-5019, CNCM
I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023 and any combination
thereof.
29. The method according to claim 14, wherein said method reduces
weight gain of the subject.
30. The method according to claim 14, wherein said method improves
glucose tolerance, insulin sensitivity and fatty liver disease in
the subject.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of medicine, in
particular of the treatment of metabolic syndrome and the
associated disorders.
BACKGROUND OF THE INVENTION
[0002] The metabolic syndrome is a cluster of the most dangerous
heart attack and diabetes risk factors. This represents a major
health problem because a quarter of the world's adults have
metabolic syndrome. People with metabolic syndrome are twice as
likely to die from, and three times as likely to have a heart
attack or stroke compared with people without the syndrome. People
with metabolic syndrome have a five-fold greater risk of developing
type 2 diabetes.
[0003] Aryl-hydrocarbon receptor (AhR) is a ligand-activated
nuclear receptor/transcription factor that regulates genes involved
in toxicant metabolism and provides a major defense to
environmental exposures. The AhR can be activated by dietary
components such as fats and fat derivatives, and there is evidence
linking the activated AhR to major diseases, including obesity (La
Merill et al, 2009, Environ Health Perspect, 117, 1414-1419).
[0004] More recent publications suggest that AhR activity would be
associated obesity and suggest that its inhibition would be
beneficial against obesity.
[0005] Several studies have examined the AhR and fat metabolism
using a model system comparing functional AhR signaling to one that
is AhR deficient. Xu at al (2015, Int J Obes, 39, 1300-1309)
concluded that AhR deficiency protected against HFD (high Fat
Diet)-induced obesity, hepatic steatosis, insulin resistance and
inflammation, and preserved insulin signaling in major metabolic
tissues. The authors suggested to target AhR for the development of
pharmaceuticals, independent of food intake, to combat obesity and
diabetes.
[0006] Kerley-Hamilton et al (2012, Environ Health Perspect, 120,
1252-1259) studied a model system with two different functional
AhR, namely with high-affinity AhR and low-affinity AhR. They
showed that mice with high-affinity AhR are more susceptible to
obesity than mice with low-affinity AhR when fed Western diet.
Therefore, the authors suggested to use of AhR antagonists for the
treatment of obesity.
[0007] Park et al (2013, Biofactor, 39, 494-504) concluded that
circulating AhR ligands may directly reduce mitochondrial function
in tissues, leading to weight gain, glucose intolerance, and
metabolic syndrome.
[0008] More recently, Moyer et al (2016, Toxicol Appl Pharmacol,
300:13-24) investigated into whether inhibition of the AHR prevents
Western diet-based obesity and came to the conclusion that AHR
antagonists .alpha.-naphthoflavone and CH-223191 significantly
reduce obesity and adiposity and ameliorates liver steatosis in
male C57Bl/6J mice fed a Western diet.
[0009] The metabolic syndrome remains a major issue for the health
and it exists a strong need of any means suitable for fighting such
a plague.
SUMMARY OF THE INVENTION
[0010] Surprisingly, the inventors observed that animal models of
metabolic syndrome (high fat diet (HFD)-induced metabolic syndrome
or leptin deficient mice (ob/ob mice)) are associated with a
decreased AhR agonist activity of their gut microbiota and the
administration of AhR agonist, either via a pharmacological
strategy or a via an intestinal bacterium naturally producing AhR
agonist, reduces the weight gain, and improves glucose tolerance,
insulin sensitivity and fatty liver disease. In addition, they
observed that, in human, the AhR agonist activity of the gut
microbiota is inversely correlated with the metabolic syndrome.
[0011] Therefore, the present invention relates to a bacterial
probiotic producing an aryl hydrocarbon receptor (AhR) agonist or
an aryl hydrocarbon receptor (AhR) agonist for use for the
preventive or curative treatment of metabolic syndrome and the
associated disorders in a subject.
[0012] In particular, the associated disorders are selected from
the group consisting of cardiovascular disease, in particular
coronary heart disease, especially heart attack and stroke, insulin
resistance, glucose intolerance, type 2 diabetes, fatty liver
disease, in particular non-alcoholic fatty liver disease and
non-alcoholic steatohepatitis, and lipodystrophy.
[0013] In one embodiment, the subject presents a decreased AhR
activity, more particularly a decreased AhR activity of the gut
microbiota.
[0014] In one embodiment, the AhR agonist can be selected, but is
not limited to, from the group consisting of indoles derivatives,
tryptophan catabolites of the microbiota, kynurenine, kynurenic
acid, indole-3-aldehyde (IAld), tryptamine, indole 3-acetate,
3-indoxyl sulfate, 6-formylindolo(3,2-b)carbazole (FICZ),
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tryptophan derivatives,
flavonoids, biphenyls, AhR modulator (SAhRM), diindolylmethane
(DIM), methyl-substituted diindolylmethanes, dihalo- and dialkylDIM
analogs, mexiletine, polycyclic aromatic hydrocarbon (PAH),
polychlorinated biphenyl (PCB), .beta.-naphthoflavone (.beta.NF),
5,6 benzoflavone (5,6 BZF), 3-indoxyl-sulfate
(13S),1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazol-
yl)ethanone hydrobromide (Pifithrin-.alpha. hydrobromide),
(2'Z,3'E)-6-Bromo-1-methylindirubin-3'-oxime (MeB10),
5-hydroxy-7-methoxyflavone, 7-methoxyisoflavone, 6-methylflavone,
3-hydroxy-6-methylflavone, pinocembrin (5,7-dihydroxyflavanone) and
7,8,2'-trihydroxyflavone, 1,4-dihydroxy-2-naphthoic acid (DHNA),
SU5416, CB7950998, Nimidipine, Flutamide, Atorvastatin,
Leflunomide, Ginseng and natural AhR Agonists (NAhRAs).
[0015] In one embodiment, the bacterial probiotic producing an AhR
agonist is a bacterium naturally producing an AhR agonist or a
modified bacterium producing an AhR agonist, such as Allobaculum
such as Allobaculum stercoricanis, Lactobacillus such as
Lactobacillus reuteri, Lactobacillus taiwanensis, Lactobacillus
johnsonii, Lactobacillus animalis, Lactobacillus murinus,
Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus
bulgaricus, and Lactobacillus delbrueckii subsp. Bulgaricus, the
genus Adlercreutzia, the phylum Actinobacteria, lactic acid
bacterium, Streptococcus thermophilus, Bifidobacterium, Propionic
acid bacterium, Bacteroides, Eubacterium, anaerobic Streptococcus,
Anaerostipes such as Anaerostipes hadrus, Anaerostipes caccae, and
Anaerostipes butyraticus, Enterococcus, Ruminococcus gnavus,
Faecalibacterium prausnitzii, or Escherichia coli. In a preferred
embodiment, the bacterial probiotic is an Allobaculum. In an
alternative preferred embodiment, the bacterial probiotic is a
Lactobacillus, preferably selected in the group consisting of
Lactobacillus reuteri, Lactobacillus taiwanensis, Lactobacillus
animalis, Lactobacillus murinus, Lactobacillus salivarius,
Lactobacillus gasseri, Lactobacillus bulgaricus, and Lactobacillus
delbrueckii subsp. Bulgaricus, more preferably of Lactobacillus
reuteri, Lactobacillus taiwanensis, Lactobacillus animalis,
Lactobacillus murinus, Lactobacillus salivarius, and Lactobacillus
gasseri, still more preferably Lactobacillus reuteri, Lactobacillus
taiwanensis, Lactobacillus animalis, and Lactobacillus murinus.
[0016] Preferably, the bacterial probiotic is selected from the
group consisting of the strain CNCM I-5019, CNCM I-5020, CNCM
I-5021, CNCM I-5022, CNCM I-5023, Ruminococcus gnavus ATCC29149,
Lactobacillus salivarius DSM20555, Lactobacillus reuteri DSM20016,
Lactobacillus gasseri DSM20243T, Faecalibacterium prausnitzii
A2-165, Escherichia coli MG1665, Anaerostipes hadrus DSM3319,
Anaerostipes caccae DSM14662, Anaerostipes butyraticus DSM22094,
Allobaculum stercoricanis DSMZ13633 and combinations thereof. In a
preferred embodiment, the bacterial probiotic is selected from the
group consisting of bacterial probiotics available under CNCM
deposit numbers CNCM I-5019, CNCM I-5020, CNCM I-5021, CNCM I-5022,
CNCM I-5023 and any combination thereof. Alternatively, the
bacterial probiotic is Lactobacillus delbrueckii subsp. Bulgaricus,
in particular OLL1181 strain.
[0017] In one embodiment, the bacterial probiotic is to be
administered by oral or rectal route. When an AhR agonist is used,
the AhR agonist is to be administered by enteral or parenteral
route, preferably be enteral route.
[0018] In addition, the present invention also relates to a
bacterial probiotic selected from the group consisting of bacterial
probiotics available under CNCM deposit numbers CNCM I-5019, CNCM
I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023 and any combination
thereof for use for the preventive or curative treatment of
obesity.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The inventors demonstrated that a metabolic syndrome induced
by high fat diet (HFD) or genetically induced (ob/ob mice) leads to
an impaired ability of the intestinal microbiota to produce AhR
agonists. Correcting this defect by stimulation of AhR has a
protective effect on weight gain, glycemic control, fatty liver
disease and intestinal inflammation induced by HFD diet. These
results are relevant for human as the same impaired ability of the
microbiota to produce AhR agonists has been observed in obese
patients and/or patients suffering from metabolic syndrome.
Therefore, the stimulation of AhR could be an interesting
preventive or curative treatment of metabolic syndrome and the
associated disorders.
[0020] The invention relates to an AhR agonist or a bacterial
probiotic producing an AhR agonist for use for the preventive or
curative treatment of metabolic syndrome and the associated
disorders. It also relates to the use of a bacterial probiotic
producing an AhR agonist or an AhR agonist for the manufacture of a
medicament for the preventive or curative treatment of metabolic
syndrome and the associated disorders in a subject. It finally
relates to a method for preventive or curative treatment of
metabolic syndrome and the associated disorders in a subject
comprising administering to the subject a bacterial probiotic
producing an AhR agonist or an AhR agonist.
[0021] Metabolic Syndrome and Associated Disorders
[0022] The metabolic syndrome is defined by a clustering of at
least three of the five following medical conditions: [0023]
Abdominal (central) obesity; [0024] Elevated blood pressure; [0025]
Elevating fasting plasma glucose; [0026] High serum triglycerides;
and [0027] Low high-density lipoprotein (HDL) levels.
[0028] It is important to note that obesity doesn't equate to
metabolic syndrome. Patients who are of normal weight may also have
the metabolic syndrome and, on the opposite, obese people may not
have a metabolic syndrome. Indeed, as mentioned above, the
metabolic syndrome is established if the obesity is an abdominal
obesity and if at least two other medical conditions are
observed.
[0029] According to the International Diabetes Federation, a
consensus worldwide definition of the metabolic syndrome (2006) is:
Central obesity (defined as waist circumference with
ethnicity-specific values) and any two of the following: the
abdominal (central) obesity: [0030] Elevated blood pressure (BP):
systolic BP >130 or diastolic BP >85 mm Hg, or treatment of
previously diagnosed hypertension; [0031] Elevating fasting plasma
glucose (FPG): >100 mg/dL (5.6 mmol/L), or previously diagnosed
type 2 diabetes; [0032] High serum triglycerides refers to >150
mg/dL (1.7 mmol/L) or specific treatment for this lipid
abnormality. [0033] Low high-density lipoprotein (HDL) levels:
<40 mg/dL (1.03 mmol/L) in males, <50 mg/dL (1.29 mmol/L) in
females, or specific treatment for this lipid abnormality.
[0034] If BMI is >30 kg/m.sup.2, central obesity can be assumed
and waist circumference does not need to be measured.
[0035] Metabolic syndrome is associated with the risk of developing
cardiovascular diseases, in particular coronary heart disease,
especially heart attack and stroke, with insulin resistance, with
glucose intolerance, with type 2 diabetes, with fatty liver
disease, especially steatohepatitis, in particular non-alcoholic
steatohepatitis, and with lipodystrophy.
[0036] In one particular embodiment, the metabolic syndrome is not
a postmenopausal metabolic syndrome.
[0037] AhR
[0038] As used herein, the term "AhR" has its general meaning in
the art and refers to Aryl hydrocarbon receptor, a transcription
factor which is activated by diverse compounds and regulates the
expression of xenobiotic metabolism genes. Aryl hydrocarbon
receptor (AhR) is a member of the family of basic helix-loop-helix
transcription factors, the bHLH-PAS (basic
helix-loop-helix/Per-ARNT-Sim) family (Schmidt and Bradfield, 1996,
Annu Rev Cell Dev Biol. 12, 55-89; Safe S et al. 2013, Toxicol
Sci., 135, 1-16). It is described in the Uniprot database under
P35869. The sequences of reference in Genbank are the followings:
NM_001612.1 and NP 001621.4.
[0039] The term "AhR activity" has its general meaning in the art
and refers to the biological activity associated with the
activation of the AhR resulting from its signal transduction
cascade, and including any of the downstream biological effects
resulting from the binding of the candidate agent to AhR that may
be equal or higher than the biological effect resulting from the
binding of the AhR to its natural ligands.
[0040] Analyzing the AhR activation level may be assessed by any of
a wide variety of well-known methods (Lehmann et al., 1995, Journal
of Biological Chem., 270, 12953-12956; He et al., 2011, Environ
Toxicol Chem, 30, 1915-1925; and Gao et al., 2009, Anal Biochem,
393, 163-175).
[0041] Subjects
[0042] The subject is a mammal, preferably a human being. In a
particular embodiment, the subject is a man. In another particular
embodiment, the subject is a woman. In a very particular
embodiment, the subject is not a postmenopausal woman.
[0043] In on embodiment, the subject presents a decreased AhR
activity, especially in a feces sample, more particularly a
decreased AhR agonist activity of the gut microbiota.
[0044] In a particular embodiment, the activity of AhR is measured
for the subject. Preferably, the AhR activity is the activity of
the microbiota and is measured in a feces sample.
[0045] The present invention relates to a method of selecting a
subject suffering of a metabolic syndrome for a treatment according
to the present invention, wherein the subject presents a decreased
AhR activity, especially in a feces sample. More particularly, the
method comprises the steps of: i) determining the AhR agonist
activity of the microbiota in a feces sample obtained from the
subject, ii) comparing the level determined at step i) with a
predetermined reference value, and iii) selecting the subject as
suitable for the treatment when the level determined at step i) is
lower than the predetermined reference value.
[0046] In one embodiment, the AhR activation level of the
microbiota in a feces sample obtained from the subject is assessed
by cell-based assays such as described in the example, He et al.,
2011, supra and Gao et al., 2009, supra. The AhR activation level
may be assessed by luciferase activity in AhR-responsive
recombinant cells such as AhR-responsive recombinant guinea pig
(G16L1.1c8), rat (H4L1.1c4), mouse (H1L1.1c2) and human (HG2L6.1c3)
cells. The AhR activation level may also be assessed by measuring
the ability to stimulate AhR-dependent gene expression using
recombinant mouse hepatoma (Hepa1c1c7) cell-based CALUX (H1L1.1c2
and H1L6.1c2) clonal cell lines that contain a stably integrated
AhR-/dioxin-responsive element (DRE)-driven firefly luciferase
plasmid (pGudLuc1.1 or pGudLuc6.1, respectively) and CAFLUX
(H1G1.1c3) clonal cell lines (He et al., 2011, supra). Typically,
the AhR expression level is measured by performing the method
described in the example.
[0047] In one embodiment, the AhR activation level of the
microbiota in a feces sample obtained from the subject is assessed
by measuring tryptophan metabolism. Accordingly, the AhR activation
level may be assessed by measuring Tryptophan (Trp), kynurenine
(Kyn) and indoles derivatives indole-3-acetic acid (IAA)
concentrations (or other tryptophan metabolites), measuring
Kyn/Trp, IAA/Trp and Kyn/IAA concentrations ratios.
[0048] In one embodiment, the AhR activation level is assessed
using colon samples obtained from the subject by analyzing the
expression of AhR target genes (such as interleukins IL-22 and
IL-17), measuring IL-17.sup.+ and IL-22.sup.+ cells number,
measuring AhR and chaperone proteins heterodimerization, measuring
AhR nuclear translocation, or measuring AhR binding to its
dimerization partner (AhR nuclear translocator (ARNT)).
[0049] As used herein, the "reference value" refers to a threshold
value or a cut-off value. Typically, a "threshold value" or
"cut-off value" can be determined experimentally, empirically, or
theoretically. A threshold value can also be arbitrarily selected
based upon the existing experimental and/or clinical conditions, as
would be recognized by a person of ordinary skilled in the art. The
threshold value has to be determined in order to obtain the optimal
sensitivity and specificity according to the function of the test
and the benefit/risk balance (clinical consequences of false
positive and false negative). Typically, the optimal sensitivity
and specificity (and so the threshold value) can be determined
using a Receiver Operating Characteristic (ROC) curve based on
experimental data. Preferably, the person skilled in the art may
compare the AhR activation levels (obtained according to the method
of the invention) with a defined threshold value. In one embodiment
of the present invention, the threshold value is derived from the
AhR activation level (or ratio, or score) determined in a feces
sample derived from one or more healthy subjects, especially who
are not suffering of a metabolic syndrome. Furthermore,
retrospective measurement of the AhR activation level (or ratio, or
scores) in properly banked historical subject samples may be used
in establishing these threshold values.
[0050] In one embodiment, the subject to be treated has a High Fat
diet or a high calorie diet and not a heart-healthy diet. For
instance, the high fat diet provides more than 30% of energy as
fat.
[0051] AhR Agonist
[0052] The term "AhR agonist" has its general meaning in the art
and refers to a compound that selectively activates the AhR. The
term "AhR agonist" refers to natural AhR ligands and any compound
that can directly or indirectly stimulate the signal transduction
cascade related to the AhR. As used herein, the term "selectively
activates" refers to a compound that preferentially binds to and
activates AhR with a greater affinity and potency, respectively,
than its interaction with the other members of bHLH-PAS
transcription factors family. Compounds that prefer AhR, but that
may also activate other sub-types, as partial or full agonists are
contemplated. Typically, an AhR agonist is a small organic molecule
or a peptide.
[0053] Tests and assays for determining whether a compound is an
AhR agonist are well known by the skilled person in the art such as
described in Ji et al., 2015, Dig Dis Sci, 60, 1958-1966; Furumatsu
et al., 2011, Dig Dis Sci, 56, 2532-2544; WO 2013/171696; WO
2012/015914; U.S. Pat. No. 6,432,692. In vitro and in vivo assays
may be used to assess the potency and selectivity of the candidate
agents to induce AhR activity.
[0054] Activities of the candidate agents, their ability to bind
AhR and their ability to induce similar effects to those of indole
derivatives such as indole-3-aldehyde (IAld) or
6-formylindolo(3,2-b)carbazole (FICZ), may be tested using isolated
cells expressing AhR, AhR-responsive recombinant cells, colonic and
small intestine lamina proporia cells expressing AhR, Th17/Th22
cells, .gamma..delta.T cells, NKp46.sup.+ ILC cells, group 3 innate
lymphoid cells (ILC3s) expressing the AhR, CHO cell line cloned and
transfected in a stable manner by the human AhR or other tissues
expressing AhR.
[0055] Activities of the candidate agents and their ability to bind
to the AhR may be assessed by the determination of a Ki on the AhR
cloned and transfected in a stable manner into a CHO cell line and
measuring the expression of AhR target genes, measuring Trp, Kyn
and indoles derivatives (IAA) concentrations, measuring Kyn/Trp,
IAA/Trp and Kyn/IAA concentrations ratios, measuring IL-17.sup.+
and IL-22.sup.+ cells, measuring AhR and chaperone proteins
heterodimerization, measuring AhR nuclear translocation, or
measuring AhR binding to its dimerization partner (AhR nuclear
translocator (ARNT)) in the present or absence of the candidate
agent.
[0056] The AhR agonist activity can be for instance assess by the
expression of one or more AhR target genes, such as the AhR
repressor AHRR, and isozymes of the cytochrome P450 family 1 such
as CYP1B1, CYP1A1 and CYP1A2.
[0057] Cells, intestine cells and other tissues expressing another
receptor than AhR may be used to assess selectivity of the
candidate agents.
[0058] The AhR agonists include synthetic and naturally occurring
compounds.
[0059] In one embodiment of the invention, the agent which is an
AhR agonist may be a molecule, or a mixture of agents such
botanical extract, that directly interacts with the AhR protein.
Preferably, it induces its dissociation from the chaperone proteins
resulting in its translocation into the nucleus and dimerizing with
ARNT (AhR nuclear translocator), and leads to changes in target
genes transcription to produce a physiological effect. Reference to
"ARNT" and "aryl hydrocarbon nuclear translocator" herein includes
all mammalian versions of the protein and gene encoding the
protein. In one aspect, ARNT is human ARNT.
[0060] Agonists of AhR include halogenated aromatic hydrocarbons
(polychlorinated dibenzodioxins, dibenzofurans and biphenyls) and
polycyclic aromatic hydrocarbons (3-methylcholanthrene,
benzo-.alpha.-pyrene, benzanthracenes and benzoflavones). In
particular, they include, but are not limited to, indoles
derivatives, tryptophan catabolites such as tryptophan catabolites
of the microbiota, kynurenine, kynurenic acid, indole-3-aldehyde
(IAld), tryptamine, indole 3-acetate, 3-indoxyl sulfate,
6-formylindolo(3,2-b)carbazole (FICZ), indolo(3,2-b)carbazole
(ICZ), 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl
ester (ITE), its precursor
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate (ITC) and analogs
thereof disclosed in U.S. Pat. No. 7,419,992,
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polycyclic aromatic
hydrocarbon (PAH), polychlorinated biphenyl (PCB),
beta-naphthoflavone (BNF), 3-indoxyl-sulfate
(13S),1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazol-
yl)ethanone hydrobromide (Pifithrin-.alpha. hydrobromide),
(2'Z,3'E)-6-Bromo-1-methylindirubin-3'-oxime (MeB10), tryptophan
derivatives, flavonoids and biphenyls, and the mixtures
thereof.
[0061] Naturally occurring compounds that have been identified as
ligands of AhR include derivatives of tryptophan such as indigo dye
and indirubin, tetrapyrroles such as bilirubin, the arachidonic
acid metabolites lipoxin-A4 and prostaglandin G, modified
low-density lipoprotein and several dietary carotinoids (Denison et
al., 2002, Chem. Biol. Interact. 141, 3-24; Denison et al., 2003,
Annu. Rev. Pharmacol. Toxicol. 43, 309-334; Adachi J et al., 2001,
J. Biol. Chem., 276, 31475-1478; Sinal C J and Bend J R, 1997, Mol.
Pharmacol., 52, 590-9; Seidel S D, et al., 2001, J. Biochem. Mol.
Toxicol., 15, 187-196; McMillan B J and Bradfield C A, 2007, Proc.
Natl. Acad. Sci. U.S.A., 104, 1412-1417; Stevens et al., 2009,
Immunology., 127, 299-311).
[0062] AhR agonists are as disclosed in Bisson et al., 2009, J.
Med. Chem, 52, 5635-5641, for example, 5-hydroxy-7-methoxyflavone,
7-methoxyisoflavone, 6-methylflavone, 3-hydroxy-6-methylflavone,
pinocembrin (5,7-dihydroxyflavanone) and
7,8,2'-trihydroxyflavone.
[0063] Other examples of AhR agonists are compound VAF347
[4-(3-chlorophenyl)-N-[4-(trifluoromethyl)phenyl]pyrimidin-2-amine],
and its pro-drug version VAG539
[4-(3-chloro-phenyl)-pyrimidin-2-yl]-(4-trifluoromethyl-phenyl)-carbamic
acid 2-[(2-hydroxy-ethyl)-methyl-amino]-ethyl ester] (Lawrence B P,
2008, Blood, 112, 1158-1165).
[0064] Another example is Semaxanib (SU5416)
[3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1,3-dihydro-indole-2-one].
SU5416 was initially characterized as a potent and selective
synthetic inhibitor of VEGF receptor/pathway, but was shown to be
an AhR agonist that activates the human AhR with a potency
approaching TCDD (Mezrich J D, et al. (2012) PLoS ONE 7(9):
e44547).
[0065] In one embodiment, the compound which is a AhR agonist may
be a selective AhR modulator (SAhRM) such as diindolylmethane
(DIM), methyl-substituted diindolylmethanes, dihalo- and dialkylDIM
analogs, mexiletine, .beta.-naphthoflavone (3NF) (5,6 benzoflavone
(5,6 BZF) and moieties described, for example, in Safe et al.,
2013, Toxicol Sci., 135, 1-16; Furumatsu et al., 2011, Dig Dis Sci,
56, 2532-2544; and WO 2012/015914.
[0066] An AhR agonist also includes compounds described in WO
2012/015914 such as CB7950998.
[0067] An AhR agonist also includes natural extracts or fractions
which are activators of the AhR pathway such as
1,4-dihydroxy-2-naphthoic acid (DHNA) and natural AhR Agonists
(NAhRAs) disclosed in WO 2013/171696 and WO 2009/093207.
[0068] In a preferred embodiment, the AhR agonist is selected from
a group of approved drugs having an agonist effect on AhR and
consisting of Mexiletine, Nimidipine, Flutamide, Atorvastatin,
Leflunomide, and Ginseng (Hu W et al, 2007, Mol Pharmacol., 71,
1475-86, O'Donnell E F, et al, 2010, PLoS One, 5(10). pii: e13128;
Wang Y, et al, 2008, Eur J Pharmacol., 601, 73-78).
[0069] In one embodiment, the AhR agonists of the present invention
excludes 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid
methyl ester (ITE), its precursor
2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylate (ITC) and analogs
thereof disclosed in U.S. Pat. No. 7,419,992. In another
embodiment, the AhR agonists of the present invention excludes the
AhR agonists having an effect on the angiogenesis or the use of
such agonists in an amount having an effect on the
angiogenesis.
[0070] Microorganism Producing AhR Agonist
[0071] In one embodiment, the agent of the present invention is a
bacterial probiotic exhibiting AhR activation properties.
[0072] The term "bacterial probiotic" has its general meaning in
the art and refers to a useful microorganism that improves the
bacterial flora in the gastrointestinal tract and can bring a
beneficial action to the host, and a growth-promoting substance
therefor. The term "bacterial probiotic" also refers to a bacterium
forming the bacterial flora and a substance that promotes the
growth of such a bacterium. The term "bacterial probiotic" also
refers to a useful microorganism that can bring a beneficial action
to a host and substance produced by these microorganisms
(microorganism culture). The term "bacterial probiotic" also refers
to a dead microbial body and a microbial secretory substance.
Because of a suitable enteric environment being formed and the
action being independent of differences in enteric environment
between individuals, the probiotic is preferably a living
microbe.
[0073] The term "bacterial probiotic exhibiting AhR activation
properties" has its general meaning in the art and relates to a
probiotic which can activate the AhR. The term "bacterial probiotic
exhibiting AhR activation properties" also relates to a probiotic
capable of activating the AhR or having AhR activating potency. The
term "AhR activation properties" means potency in being able to
activate a signaling pathway that is initiated by AhR activation,
and may involve any kind of activating mechanism. Therefore, it is
not always necessary for a microbial body itself to be an AhR
ligand, and for example a secretory substance produced by a microbe
may have AhR-activating potency, or the AhR may be activated by a
dead microbial body or homogenate thereof. A growth-promoting
substance having AhR-activating potency includes a case in which
the substance itself has AhR-activating potency and also a case in
which the substance itself does not have AhR-activating potency but
it promotes growth of a bacterium having AhR-activating potency.
Therefore, when a "microorganism" or "bacterium" is referred to or
a specific microbe is referred to in the present invention, they
include not only a living microbe but also a dead microbial body or
homogenate thereof and a culture of said microbe or a secretory
substance. However, it is preferably a microbial body itself such
as a living microbe or a dead microbial body or homogenate thereof,
and from the viewpoint of being capable of forming bacterial flora
in the gastrointestinal tract, it is more preferably a living
microbe (US 2013/0302844).
[0074] Bacterial probiotics include, but are not limited to,
bacterium exhibiting naturally AhR activation properties or
modified bacterium exhibiting AhR activation properties such as
Allobaculum, Lactobacillus reuteri, Lactobacillus taiwanensis,
Lactobacillus johnsonii, Lactobacillus animalis, Lactobacillus
murinus, the genus Adlercreutzia, the phylum Actinobacteria, lactic
acid bacterium, Lactobacillus bulgaricus, Streptococcus
thermophilus, Bifidobacterium, Propionic acid bacterium,
Bacteroides, Eubacterium, anaerobic Streptococcus, Enterococcus,
Lactobacillus delbrueckii subsp. Bulgaricus, Escherichia coli,
other intestinal microorganisms and probiotics described for
example in US 2013/0302844.
[0075] In a preferred embodiment, the bacterial probiotic is an
Allobaculum. In an alternative preferred embodiment, the bacterial
probiotic is a Lactobacillus, preferably selected in the group
consisting of Lactobacillus reuteri, Lactobacillus taiwanensis,
Lactobacillus animalis, and Lactobacillus murinus.
[0076] In particular, the inventors have isolated bacterial
probiotics exhibiting AhR activation properties and have deposited
them at the Collection Nationale de Cultures de Microorganismes
(CNCM, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex
15, France), in accordance with the terms of Budapest Treaty. More
particularly, 5 bacterial probiotics have been deposited on Sep.
30, 2015, at the CNCM with the deposit numbers CNCM I-5019
(SB6WTD3, Lactobacillus taiwanensis), CNCM I-5020 (SB6WTD4,
Lactobacillus murinus), CNCM I-5021 (SB6WTD5, Lactobacillus
animalis), CNCM I-5022 (SB6WTF6, Lactobacillus reuteri), and CNCM
I-5023 (SB6WTG6, Lactobacillus reuteri). Accordingly, in a
preferred embodiment, the bacterial probiotic exhibiting AhR
activation properties is selected from the group consisting of the
strain CNCM I-5019, CNCM I-5020, CNCM I-5021, CNCM I-5022, and CNCM
I-5023, and combinations thereof.
[0077] In a preferred embodiment, the bacterial probiotic is
capable of producing an AhR agonist. Bacterial probiotics include,
but are not limited to, bacterium naturally producing an AhR
agonist or modified bacterium producing an AhR agonist. Such
bacterial probiotic can be selected from the group consisting of
Allobaculum such as Allobaculum stercoricanis, Lactobacillus such
as Lactobacillus reuteri, Lactobacillus taiwanensis, Lactobacillus
johnsonii, Lactobacillus animalis, Lactobacillus murinus,
Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus
bulgaricus, and Lactobacillus delbrueckii subsp. Bulgaricus, the
genus Adlercreutzia, the phylum Actinobacteria, lactic acid
bacterium, Streptococcus thermophilus, Bifidobacterium, Propionic
acid bacterium, Bacteroides, Eubacterium, anaerobic Streptococcus,
Anaerostipes such as Anaerostipes hadrus, Anaerostipes caccae, and
Anaerostipes butyraticus, Enterococcus, Ruminococcus gnavus,
Faecalibacterium prausnitzii, Escherichia coli, other intestinal
microorganisms and probiotics described for example in US
2013/0302844. In one embodiment, such bacterial probiotics include,
but are not limited to, bacterium naturally producing an AhR
agonist or modified bacterium producing an AhR agonist. Bacterial
probiotic can be selected from the group consisting of Allobaculum
such as Allobaculum stercoricanis, Lactobacillus such as
Lactobacillus reuteri, Lactobacillus taiwanensis, Lactobacillus
animalis, Lactobacillus murinus, Lactobacillus salivarius,
Lactobacillus gasseri, Anaerostipes such as Anaerostipes hadrus,
Anaerostipes caccae, and Anaerostipes butyraticus, Ruminococcus
such as Ruminococcus gnavus, Faecalibacterium such as
Faecalibacterium prausnitzii, and Enterobacteriaceae such as
Escherichia coli. More specifically, such bacterial probiotic can
be selected from the group consisting of the strain CNCM I-5019,
CNCM I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023, Ruminococcus
gnavus ATCC29149, Lactobacillus salivarius DSM20555, Lactobacillus
reuteri DSM20016, Lactobacillus gasseri DSM20243T, Faecalibacterium
prausnitzii A2-165, Escherichia coli MG1665, Anaerostipes hadrus
DSM3319, Anaerostipes caccae DSM14662, Anaerostipes butyraticus
DSM22094, Allobaculum stercoricanis DSMZ13633 and combinations
thereof. In a particular embodiment, bacterial probiotic can be
selected from the group consisting of the strain CNCM I-5019, CNCM
I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023, and combinations
thereof. The bacteria have been shown to produce AhR agonists (FIG.
19).
[0078] For instance, the patent application WO2015/025259 describes
a bacterium producing indole-3-aldehyde.
[0079] The patent application US 2013/302844 also provides
bacterial probiotics having AhR activating potency. The probiotics
is selected from the group consisting of a Lactic acid bacterium, a
Bifidobacterium, and a Propionic acid bacterium. Preferably, the
probiotics is Lactobacillus delbrueckii subsp. bulgaricus OLL1181
strain (deposited on Jul. 16, 2010, with the International Patent
Organism Depository, National Institute of Advanced Industrial
Science and Technology (Chuo No. 6, 1-1-1 Azuma, Tsukuba city,
Ibaraki prefecture, Japan 305-8566) with depository number: FERM
BP-11269).
[0080] The AhR activation level by the bacterial probiotic
exhibiting AhR activation properties or by the bacterial probiotic
producing an AhR agonist can be assessed by cell-based assays such
as described in the example, He et al., 2011, supra and Gao et al.,
2009, supra. The AhR activation level may be assessed by luciferase
activity in AhR-responsive recombinant cells such as AhR-responsive
recombinant guinea pig (G16L1.1c8), rat (H4L1.1c4), mouse
(H1L1.1c2) and human (HG2L6.1c3) cells. The AhR activation level
may also be assessed by measuring the ability to stimulate
AhR-dependent gene expression using recombinant mouse hepatoma
(Hepa1c1c7) cell-based CALUX (H1L1.1c2 and H1L6.1c2) clonal cell
lines that contain a stably integrated AhR-/dioxin-responsive
element (DRE)-driven firefly luciferase plasmid (pGudLuc1.1 or
pGudLuc6.1, respectively) and CAFLUX (H1G1.1c3) clonal cell lines
(He et al., 2011, supra). Typically, the AhR activation level is
measured by performing the method described in the example, in
particular in example 3. If the AhR agonist activity is measured by
the method of example 3, the AhR fold change is at least 2,
preferably at least 4.
[0081] In a particular embodiment, the present invention
specifically relates to a bacterial probiotic selected from the
group consisting of the strain CNCM I-5019, CNCM I-5020, CNCM
I-5021, CNCM I-5022, and CNCM I-5023, and a combination thereof for
use for the curative or preventive treatment of obesity. It further
relates to a method for curative or preventive treatment of obesity
of a subject, comprising administering a therapeutic effective
amount of a bacterial probiotic selected from the group consisting
of the strain CNCM I-5019, CNCM I-5020, CNCM I-5021, CNCM I-5022,
and CNCM I-5023, and a combination thereof to the subject. Finally,
it relates to the use of a bacterial probiotic selected from the
group consisting of the strain CNCM I-5019, CNCM I-5020, CNCM
I-5021, CNCM I-5022, and CNCM I-5023, and a combination thereof for
the manufacture of a medicament for use for the curative or
preventive treatment of obesity. The subject is an obese having a
body mass index (BMI) higher than 30, preferably higher than 35,
more preferably higher than 40, still more preferably from 40 to
50.
[0082] Optionally, the AhR agonist or bacterial probiotic
exhibiting aryl hydrocarbon receptor (AhR) activation, in
particular bacterial probiotic an AhR agonist, can be used in
combination with an additional active ingredient.
[0083] When a bacterial probiotic is used in the treatment, it can
be used in combination with other probiotics.
[0084] Pharmaceutical Composition and Administration
[0085] When the bacterial probiotics exhibiting AhR activation
properties, in particular a bacterial probiotic producing an AhR
agonist, is used for the treatment of the invention, the preferred
administration route is oral or rectal, preferable oral route.
Accordingly, in one embodiment, an oral composition comprising the
bacterial probiotic exhibiting AhR activation properties, in
particular a bacterial probiotic producing an AhR agonist, is
used.
[0086] The term "oral composition" has its general meaning in the
art and refers to any composition that can be ingested orally.
[0087] Typically, the orally ingested composition is selected from
the group consisting of a beverage or drink composition, a food
composition, a feedstuff composition and a pharmaceutical
composition.
[0088] The dosage of the AhR agonist or the bacterial probiotic
exhibiting AhR activation properties, in particular a bacterial
probiotic producing an AhR agonist, is to be appropriately adjusted
according criteria such as age, symptoms, body weight, and intended
application.
[0089] The dosage is selected such as to obtain a therapeutically
efficient amount. By therapeutically efficient amount can be
defined as the amount necessary for having an impact on one of the
five medical conditions defining the metabolic syndrome: [0090]
Abdominal (central) obesity (TOF1); [0091] Elevated blood pressure;
[0092] Elevating fasting plasma glucose; [0093] High serum
triglycerides; and [0094] Low high-density lipoprotein (HDL)
levels.
[0095] In addition or alternatively, the therapeutically efficient
amount can be evaluated by the insulin sensitivity, the glucose
tolerance, the weight gain and/or the intestinal inflammation due
to HFD. In a preferred embodiment, the therapeutically efficient
amount has an impact on several of these conditions.
[0096] For example, the amount ingested per day as the bacterial
probiotic is typically 0.01 to 100.times.10.sup.11 cells/body,
preferably 0.1 to 10.times.10.sup.11 cells/body, and more
preferably 0.3 to 5.times.10.sup.11 cells/body. Furthermore, for
example, the amount ingested per day as the bacterial probiotic is
0.01 to 100.times.10.sup.11 cells/60 kg body weight, preferably 0.1
to 10.times.10.sup.11 cells/60 kg body weight, and more preferably
0.3 to 5.times.10.sup.11 cells/60 kg body weight.
[0097] The content of the bacterial probiotic contained in the
orally ingested composition may be determined as appropriate
depending on its application form. Typically, as probiotic dry
microbial body it is for example 5 to 50 w/w %, preferably 1 to 75
w/w %, and more preferably 0.1 to 100 w/w % and 1 to 100 w/w %.
[0098] When an AhR agonist is used for the treatment of the
invention, the preferred administration route is enteral or
parenteral, preferably by oral, sublingual, subcutaneous,
intramuscular, intravenous, transdermal, local or rectal
administration, more preferably by oral administration.
[0099] Typically, the agent of the invention may be combined with
pharmaceutically acceptable excipients, and optionally
sustained-release matrices, such as biodegradable polymers, to form
therapeutic compositions.
[0100] "Pharmaceutically" or "pharmaceutically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to a mammal,
especially a human, as appropriate. A pharmaceutically acceptable
carrier or excipient refers to a non-toxic solid, semi-solid or
liquid filler, diluent, encapsulating material or formulation
auxiliary of any type.
[0101] In the pharmaceutical compositions of the present invention
for oral, sublingual, subcutaneous, intramuscular, intravenous,
transdermal, local or rectal administration, the active principle,
alone or in combination with another active principle, can be
administered in a unit administration form, as a mixture with
conventional pharmaceutical supports, to animals and human beings.
Suitable unit administration forms comprise oral-route forms such
as tablets, gel capsules, powders, granules and oral suspensions or
solutions, sublingual and buccal administration forms, aerosols,
implants, subcutaneous, transdermal, topical, intraperitoneal,
intramuscular, intravenous, subdermal, transdermal, intrathecal and
intranasal administration forms and rectal administration
forms.
[0102] Preferably, the pharmaceutical compositions contain vehicles
which are pharmaceutically acceptable for a formulation capable of
being injected. These may be in particular isotonic, sterile,
saline solutions (monosodium or disodium phosphate, sodium,
potassium, calcium or magnesium chloride and the like or mixtures
of such salts), or dry, especially freeze-dried compositions which
upon addition, depending on the case, of sterilized water or
physiological saline, permit the constitution of injectable
solutions.
[0103] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form must be sterile
and must be fluid to the extent that easy syringability exists. It
must be stable under the conditions of manufacture and storage and
must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi.
[0104] Solutions comprising agents of the invention as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms.
[0105] The agent of the invention can be formulated into a
composition in a neutral or salt form. Pharmaceutically acceptable
salts include the acid addition salts (formed with the free amino
groups of the protein) and which are formed with inorganic acids
such as, for example, hydrochloric or phosphoric acids, or such
organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0106] The carrier can also be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetables oils. The proper
fluidity can be maintained, for example, by the use of a coating,
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. The
prevention of the action of microorganisms can be brought about by
various antibacterial and antifungal agents, for example, parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In
many cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. Prolonged absorption of the
injectable compositions can be brought about by the use in the
compositions of agents delaying absorption, for example, aluminium
monostearate and gelatin.
[0107] Sterile injectable solutions are prepared by incorporating
the active agents in the required amount in the appropriate solvent
with several of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0108] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms, such as the type of injectable
solutions described above, but drug release capsules and the like
can also be employed.
[0109] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. Some variation in dosage
will necessarily occur depending on the condition of the subject
being treated. The person responsible for administration will, in
any event, determine the appropriate dose for the individual
subject.
[0110] In addition to the agents of the invention formulated for
parenteral administration, such as intravenous or intramuscular
injection, other pharmaceutically acceptable forms include, e.g.
tablets or other solids for oral administration; liposomal
formulations; time release capsules; and any other form currently
used.
BRIEF DESCRIPTION OF THE FIGURES
[0111] FIG. 1. AhR agonist reversed HFD-induced IL-22 deficiency.
(a) Fecal AhR activity from indicated mice fed with either CD or
HFD diet. Data are shown as mean.+-.SEM (n=5-10/group).
Quantification of (b) Il22 (c) Reg3b and (d) Reg3g mRNA transcripts
by RT-qPCR method in ileum and colon of CD- and HFD-fed mice
treated with FICZ or vehicle (DMSO). In b-d, data were quantified
using .DELTA..DELTA.Ct with Gapdh as internal control and CD-DMSO
group as a calibrator, and expressed as mean.+-.SEM (5-8/group). In
a-d, statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then multiple
comparison test using one-way analysis of variance (ANOVA) followed
by Bonferroni post-hoc test or Kruskal-Wallis test followed by
Dunn's post-hoc test.
[0112] FIG. 2. Defective AhR agonist activity of the gut microbiota
in metabolic syndrome in human. In human, AhR agonist activity of
the gut microbiota is negatively correlated with body mass index
(BMI) (A), and is lower in patients with diabetes (B) or high blood
pressure (HBP) (C). In all panels, *P<0.05, ** or
.sup..dagger..dagger.P<0.001, and *** or
.sup..dagger..dagger..dagger.P<0.0001, two-tailed Student's
t-test in panels. Spearman correlation used in panel A.
[0113] FIG. 3. AhR agonist reduced HFD-induced weight gain
independent of food intake. (a) Weight gain in CD- and HFD-fed mice
treated with FICZ or vehicle (DMSO). (b) Weekly food intake in CD-
and HFD-fed mice. In a-b, data are shown as mean.+-.SEM
(n=11-16/group) and statistical comparison was performed using
two-way analysis of variance followed by Bonferroni post-hoc test.
Statistical significance: *P<0.05, **P<0.01, ***P<0.001 vs
HFD-DMSO. (c) Body weight gain of mice that were fed with CD- or
HFD for 12 weeks with or without FICZ treatment. (d) Body weight
gain normalized to food intake of mice that were fed with CD- or
HFD for 12 weeks with or without FICZ treatment. In c-d,
statistical comparison was performed by first testing normality
using Kolmogorov-Smirnov test and then multiple comparison test
using one-way analysis of variance (ANOVA) followed by Bonferroni
post-hoc test or Kruskal-Wallis test followed by Dunn's post-hoc
test.
[0114] FIG. 4. AhR agonist improved HFD-induced glucose tolerance.
(a) Oral glucose tolerance test on mice fed with either CD or HFD
with or without FICZ treatment. Data are shown as mean.+-.SEM
(n=11-16/group) and statistical comparison was performed using
two-way analysis of variance followed by Bonferroni post-hoc test.
Statistical significance: *P<0.05, **P<0.01, ***P<0.001 vs
HFD-DMSO (b) Area under the curve (AUC) calculation (calculated
from figure a) during oral glucose tolerance test. (c) Glucose,
insulin and homeostatic model assessment (HOMA) calculation after 6
h of fasting and 30 min after oral glucose challenge. In b-c, data
shown are expressed as mean.+-.SEM (n=11-16/group) and statistical
comparison was performed by first testing normality using
Kolmogorov-Smirnov test and then multiple comparison test using
one-way analysis of variance (ANOVA) followed by Bonferroni
post-hoc test or Kruskal-Wallis test followed by Dunn's post-hoc
test.
[0115] FIG. 5. AhR agonist prevented HFD-induced dysregulated
immune response in spleen. Quantification of (a) IFN-.gamma. (b)
TNF-.alpha. (c) IL-17.alpha. and (d) IL17f cytokine production by
anti-CD3/anti-CD28-stimulated splenic cells from CD- and HFD-fed
mice treated with or without FICZ. Data shown are expressed as
mean.+-.SEM (5-8/group). Statistical comparison was performed by
first testing normality using Kolmogorov-Smirnov test and then
multiple comparison test using one-way analysis of variance (ANOVA)
followed by Bonferroni post-hoc test or Kruskal-Wallis test
followed by Dunn's post-hoc test.
[0116] FIG. 6. AhR agonist prevented HFD-induced dysregulated
mucosal immune response. (a) Quantification of IL-22+ cells
isolated from small intestine (SI) and colon lamina propria (LP).
Cells were gated on total live cells. (b) Quantification of IL-22+
cells within ILC population. Cells were gated on no T-cells,
B-cells, dendritic cells and monocytes population. (c)
Quantification of lamina propria (LP) and intraepithelial
lymphocytes (IEL) IFN-.gamma.+ cells isolated from small intestine
(SI) and colon. Cells were gated on total live cells. Data shown
are expressed as mean.+-.SEM (2-6/group). Statistical comparison
was performed by first testing normality using Kolmogorov-Smirnov
test and then multiple comparison test using one-way analysis of
variance (ANOVA) followed by Bonferroni post-hoc test or
Kruskal-Wallis test followed by Dunn's post-hoc test.
[0117] FIG. 7. Diet- and genetically-induced metabolic syndrome is
associated with altered microbiota composition and impaired
microbiota-driven AhR activation. (a) Quantification of fecal AhR
activity of mice (left) and rats (middle) fed with CD or HFD for 12
weeks, and ob/ob and wild-type (WT) mice fed with CD at 6 weeks of
age. (b) Fecal concentration of indole acetic acid (IAA) and
kynurenine of the indicated mice. (c) PCoA plot of fecal microbiota
of mice fed with CD or HFD after 12 weeks. (d) Bar graph of
bacterial abundance in family level of CD- or HFD-fed mice. For
figure a-b, statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then ANOVA or
Kruskal-Wallis test with Bonferroni or Dunn's post hoc test.
[0118] FIG. 8. HFD-fed mice showed defective tryptophan metabolism
by microbiota and metabolic host dysfunctions. (a) Concentration of
tryptophan in the colon mucosa of mice fed with CD or HFD. (b) Body
weight gain and (c) weekly food intake of mice (*p<0.05,
n=5/group). (d) Blood glucose, (e) insulin, (f) homeostatic model
assessment-insulin resistance (HOMA-IR) after 6 h of fasting. (g)
Blood glucose level before and after oral glucose tolerance
challenge (OGGT; *p<0.05, n=5/group). (h) Area under the curve
(AUC) of OGGT. (i) Blood glucose level before and after insulin
tolerance test (ITT; n=5/group). (j) Area under the curve (AUC) of
OGGT. (k) Lipid area, calculated as % area of interest (AOI), in
H&E stained liver cross-sections. (l) Liver triglycerides after
6 h of food deprivation. Statistical comparison was performed by
first testing normality using Kolmogorov-Smirnov test and then
unpaired t-test or Mann-Whitney test.
[0119] FIG. 9. Treatment with AhR agonist or supplementation with
high AhR ligand-producing bacteria alleviates both diet- and
genetically-induced metabolic impairments. (a) Fasting homeostatic
model assessment-insulin resistance (HOMA-IR) of CD- and HFD-fed
mice treated with Ficz or vehicle (DMSO). (b) Blood glucose level
before and after oral glucose tolerance challenge of CD- and
HFD-fed mice treated with Ficz or vehicle (OGGT, *p<0.05 vs HFD,
n=12-20/group). (c) Lipid area, calculated as % area of interest
(AOI), in liver cross-sections of CD- and HFD-fed mice treated with
Ficz or vehicle. (d) Representative pictures of H&E-stained
liver sections from CD- and HFD-fed mice treated with Ficz or
vehicle. (e) Fasting glucose level of ob/ob mice treated with Ficz
or vehicle. (f) Glucose level before and after OGGT of ob/ob mice
treated with Ficz or vehicle (*p<0.05, n=10-21/group). (g) Lipid
area in liver cross-sections of ob/ob mice treated with Ficz or
vehicle. (h) Representative pictures of H&E-stained liver
sections from ob/ob mice treated with Ficz or vehicle. (i)
Quantification of fecal AhR activity of mice fed with CD or HFD
supplemented with L. reuteri or vehicle. (j) Fecal concentration of
IAA and kynurenine of the indicated mice. Fasting homeostatic model
assessment-insulin resistance (HOMA-IR) of CD- and HFD-fed mice
supplemented with L. reuteri or vehicle. (k) Blood glucose level
before and after OGGT of CD- and HFD-fed mice supplemented with L.
reuteri or vehicle (*p<0.05, OGGT, n=8/group). (l)
Representative pictures of H&E-stained liver sections from CD-
and HFD-fed mice supplemented with L. reuteri or vehicle. (m) Lipid
area in liver cross-sections of CD- and HFD-fed mice supplemented
with L. reuteri or vehicle. For all data, statistical comparison
was performed by first testing normality using Kolmogorov-Smirnov
test and then ANOVA or Kruskal-Wallis test with Bonferroni or
Dunn's post hoc test.
[0120] FIG. 10. Treatment with AhR agonist Ficz attenuated
HFD-induced metabolic dysfunction. (a) Body weight gain
(n=10/group). (b) Blood glucose and (c) insulin after 6 h of
fasting. (d) Area under the curve (AUC) of OGGT. Refer to FIG. 9b
for the OGGT figure. (e) Blood glucose level before and after
insulin tolerance test (ITT; *p<0.05, n=6-10/group). (f) AUC of
ITT. Concentration of (g) alanine transaminase (ALT), (h) aspartate
transaminase (AST) and (i) total cholesterol from the serum of
indicated mice. (j) Liver triglycerides after 6 h of food
deprivation. Statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0121] FIG. 11. Treatment with AhR agonist Ficz did not improve
fecal AhR activity but attenuated HFD-associated defective
intestinal AhR activation and Il22 expression. (a) AhR activity of
stools from mice fed with CD or HFD for 12 weeks and treated with
AhR agonist Ficz or vehicle. Transcript expression of (b) Cyp1a1
(c) Il22, (d) Reg3g and (e) Reg3b in different intestinal segments
of indicated mice (n=5-12/group). Statistical comparison was
performed by first testing normality using Kolmogorov-Smirnov test
and then ANOVA or Kruskal-Wallis test with Bonferroni or Dunn's
post hoc test.
[0122] FIG. 12. Treatment with AhR agonist Ficz reduced features of
metabolic syndrome in ob/ob mice. (a) Body weight gain
(n=10-15/group). (b) Insulin and (c) homeostatic model
assessment-insulin resistance (HOMA-IR) after 6 h of fasting. (d)
Area under the curve (AUC) of OGGT. Refer to FIG. 9f for the OGGT
figure. Concentration of (e) alanine transaminase (ALT), (f)
aspartate transaminase (AST) and (g) triglycerides in the serum of
indicated mice. (h) Liver triglycerides after 6 h of food
deprivation. Statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0123] FIG. 13. Ficz treatment did not alleviate metabolic syndrome
in AhR-mice. (a) Weight gain after 12 weeks of HFD. (b) Homeostatic
model assessment-insulin resistance (HOMA-IR) after 6 h of fasting.
(c) Blood glucose level before and after OGGT (n=10/group). (d)
Area under the curve (AUC) of OGGT. (e) Blood glucose level before
and after insulin tolerance test (ITT; n=10/group). (f) AUC of ITT.
Statistical comparison was performed by first testing normality
using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0124] FIG. 14. Inoculation with high-producing AhR ligands
bacteria attenuated HFD-induced metabolic syndrome. (a) Body weight
gain (n=8/group). (b) Blood glucose and (c) insulin after 6 h of
fasting. (d) Area under the curve (AUC) of OGGT. Refer to FIG. 9k
for the OGGT figure. (e) Blood glucose level before and after
insulin tolerance test (ITT; n=8/group). (f) AUC of ITT.
Concentration (g) alanine transaminase (ALT), (h) aspartate
transaminase (AST) and (i) triglycerides in the serum of indicated
mice. Statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0125] FIG. 15. Treatment with AhR agonist or inoculation with high
AhR ligand producing improves HFD-induced intestinal barrier
dysfunction and impaired incretin secretion. Translocation of (a)
LPS, (b) sulfonic acid (FS4) and (c) dextran (ARD4) across mucosa
in different intestinal segments (n=3-5/group). (d-e)
Transepithelial resistance (TER) or fluorescein-labeled dextran
(FD4) of Caco-2 cells treated with TNF-.alpha. or vehicle in the
presence of Ficz or vehicle. Data represents one independent
experiment. (f) Concentration of soluble CD14 (sCD14) in the serum
of indicated mice. (g) TNF-.alpha. and (h) IFN-.gamma. production
of spleen cells after stimulation with PMA and ionomycin. (i)
Expression of proglucagon in different intestinal segments of
indicated mice (n=6-8/group). (j) Concentration of total GLP-1 in
the serum of indicated mice. (k) Quantification of GLP-1 secretion
by GLUTag cells after stimulation with Ficz and forskolin (positive
control) in the presence or absence of AhR antagonist (CH223191).
Data represents one independent experiment. For all data,
statistical comparison was performed by first testing normality
using Kolmogorov-Smirnovtest and then ANOVA or Kruskal-Wallis test
with Bonferroni or Dunn's post hoc test.
[0126] FIG. 16. Microbiota of individuals with metabolic syndrome
display reduced AhR activation and lower AhR agonists
concentration. (a) Quantification of fecal AhR activity of
individuals with low and high body mass index (BMI). (b) Spearman
correlation of stool AhR activation and body mass index (BMI).
(c-d) Quantification of fecal AhR activity of individuals with type
2 diabetes (T2D) and high blood pressure (HBP) compared to healthy
subjects. (e) Total concentration of 4 AhR agonist (IAA, indole,
3-methyl-indole and tryptamine) from feces of individuals with low
and high BMI. (f) Spearman correlation of stool AhR agonist
concentration and BMI. (g-h) AhR agonist concentration from feces
of individuals with T2D and compared to healthy subjects. (i)
Kyrunenine concentration from feces of individuals with low and
high BMI. (j) Spearman correlation of stool kyrunenine
concentration and BMI. (k-l) Kyrunenine concentration from feces of
individuals with T2D and compared to healthy subjects. For all
data, statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then ANOVA or
Kruskal-Wallis test with Bonferroni or Dunn's post hoc test.
[0127] FIG. 17. Microbiota of obese individuals displayed lower
levels of microbiota-derived AhR agonists. Concentration of (a)
indole acetic acid (IAA), (b) indole, (c) 3-methyl indole and (d)
Tryptamine from feces of individuals with low and high BMI. (f)
Spearman correlation of stool AhR agonist concentration and BMI.
Statistical comparison was performed by first testing normality
using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0128] FIG. 18. Microbiota from individuals with metabolic syndrome
showed lower AhR activity AhR agonist. Stools from individuals
possessing one or more metabolic risk factors showed lower (a) AhR
activity, higher (b) kyrunenine and increased level of (c) AhR
agonists. Statistical comparison was performed by first testing
normality using Kolmogorov-Smirnov test and then unpaired t-test or
Mann-Whitney test.
[0129] FIG. 19. AhR activity of bacterial strains.
EXAMPLES
Example 1
[0130] The inventors explored the role of aryl hydrocarbon receptor
(AhR) in modulating high-fat diet (HFD)-induced obesity and insulin
resistance. For this, they maintained mice on standard control diet
(CD) or high-fat diet (HFD; milk-derived fat) for twelve weeks.
They observed a decrease in AhR activity in the feces of mice fed a
HFD diet. Moreover, treating the mice with 6-Fomylindolo-(3,2-b)
carbazole (FICZ), a potent AhR agonist, reduced the weight gain and
markedly improved glucose tolerance in HFD-fed mice. This study
indicates a critical role of AhR activation in the regulation of
high-fat diet induced obesity, insulin resistance and dysregulated
immune response.
[0131] Results
[0132] Analysis of AhR Activity in HFD-Fed Mice.
[0133] After 12 weeks of CD or HFD, AhR activity in the feces was
analyzed using a luciferase reporter-based assay (Lamas et al,
2016). Ileal and colonic expressions of AhR-regulated genes were
similarly evaluated. As shown in the FIG. 1, the AhR fecal activity
of HFD-DMSO mice was significantly reduced compared to CD-DMSO
mice. This was associated with a lower expression of AhR-related
genes, such as the cytokine Il22 and the antimicrobial Reg3g and
Reg3b. Intraperitoneal injection of FICZ, an AhR agonist, rescued
Il22 and Reg3g expression in colon and ileum of mice fed a HFD diet
without changing the fecal AhR activity.
[0134] Reduced AhR Activity in the Microbiota of Human Patients
with Metabolic Syndrome
[0135] To assess whether the phenomenon observed in mice have a
clinical relevance in humans, the inventors analyzed fecal samples
from a cohort of patients in consulting in cardiology for their
ability to activate AhR. As shown in the FIG. 2, they observed a
correlation between AhR activity and (i) the body mass index (BMI),
(ii) blood pressure and (iii) diabetes status. The results suggest
that the microbiota of obese patients and/or patients with
metabolic syndrome have an impaired ability to produce AhR ligands
which could be involved in the pathogenesis of metabolic
syndrome.
[0136] Effect of AhR Activation on Weight Gain and Food Intake.
[0137] Food consumption and weight gain were recorded weekly. All
HFD-fed mice exhibit higher weight gain compared to mice fed a CD
diet. As shown in FIG. 3, HFD-fed mice treated with FICZ showed
significantly lower weight gain compared to untreated HFD-fed mice.
During the course of the experiment, no significant difference in
food consumption was detected between FICZ treated and untreated
HFD-mice, suggesting that lower weight gain in HFD-FICZ group was
independent of reduced food intake by mice.
[0138] Effect of AhR Activation on HFD-Induced Glucose Tolerance
and Insulin Sensitivity.
[0139] After 11-weeks of CD or HFD, glucose homeostasis was
evaluated using the oral glucose tolerance test (OGGT) method. In
brief, after 6 hours of fasting, mice were challenged orally with
glucose (2 g/kg of weight), and glucose and insulin levels were
measured regularly within the two-hour period after challenge. As
shown in FIG. 4, HFD-fed mice, regardless of treatment, showed
higher fasting glucose and insulin levels compared to CD-fed mice.
However, HFD-FICZ mice showed better glucose tolerance and insulin
sensitivity after glucose challenge compared to non-treated HFD-fed
mice. Overall, the results showed that activating AhR axis, using
FICZ, rectified HFD-induced glucose tolerance.
[0140] Effect of AhR Activation on HFD-Induced Systemic Immune
Response.
[0141] Cytokine production of splenic cells from CD- or HFD-fed
mice was measured after in vitro anti-CD3 and anti-CD28 stimulation
using cytokine multiplex assay. As shown in FIG. 5, HFD-DMSO mice
showed higher IFN-.gamma., TNF-.alpha., IL-17a and IL17f production
compared to CD-DMSO mice. FICZ treatment in HFD-fed mice rescued
the production of this cytokines reaching the level of CD-DMSO mice
showing that, FICZ reversed the HFD-induced dysregulated systemic
immune response.
[0142] Effect of AhR Activation on HFD-Induced Mucosal Immune
Response.
[0143] Lamina propria (LP) and intestinal epithelial cells from
small intestine (SI) and colon were isolated and stimulated with
phorbol 12-myristate 13-acetate (PMA) and ionomycin for 4 h. IL-22+
and IFN-.gamma.+ cells were quantified by flow cytometry technique.
As shown in FIG. 6, SI and colon cells of HFD-DMSO mice showed
lower total IL-22 production compared to CD-DMSO mice. The
percentage of innate lymphoid cells (ILC) producing IL-22 was also
reduced in colon LP of HFD-DMSO mice. HFD was associated with
increased production of IFN-.gamma. by SI LP cells and intestinal
epithelial lymphocytes (IEL). FICZ treatment reversed the
HFD-induced SI LP and colonic innate lymphoid cells IL-22
deficiency. Furthermore, AhR activation by FICZ reduced the
HFD-induced IFN-.gamma. production in the small intestine.
[0144] Conclusion
[0145] This work shows that a HFD diet induces an alteration in the
ability of the intestinal microbiota to produce AhR agonists,
leading to a defect in IL-22 pathway. Activating the AhR machinery
is effective in correcting these defects and confers a protective
effect on weight, glycemic control and intestinal inflammation
induced by HFD diet.
[0146] The present results have a relevant translational impact for
humans, as impaired ability to produce AhR ligands is observed in
obese patients and/or patients suffering from metabolic syndrome.
Two studies have suggested that AhR activation is deleterious in
metabolic syndrome (Kerley-Hamilton et al Environ Health Perspect
2012; Xu et al Int J Obes (Lond), 2015). However these two studies,
were conducted with AhR knockout mice (or mice with a diminished
sensitivity in a comprehensive manner with respect to AhR). On the
contrary, the inventors' work suggests that a defect of AhR ligands
in the intestine (without pre-judging the systemic effect) is
pathogenic for the development of metabolic syndrome.
[0147] Materials and Methods
[0148] Mice and Treatments.
[0149] C57BL/6J mice (Janvier) were maintained under specific
pathogen free (SPF) conditions. All mice were males and 4 weeks of
age at the start of the experiments. Mice were weight matched at
the start of the experiments. Mice were fed with irradiated control
diet (CD; Envigo MD 120508) or high-fat diet (HFD; Envigo MD
972222) containing 18% Milk-fat for 12 weeks. Fomylindolo-(3,2-b)
carbazole (FICZ) and vehicle (DMSO) were injected intraperitoneally
3 days after switching the diet to CD or HFD and then 1.times. per
week until the end of the experiment. Tissue samples were harvested
at the end of the experiment. Weekly food consumption was measured
cage-wise.
[0150] Luciferase Assay for AhR Activity Measurement.
[0151] AhR activity in the feces was measured as previously
described (Lamas et al, 2016). Briefly, frozen stool samples from
mice, healthy subjects and patients suffering from obesity or
metabolic syndrome were diluted in PBS, centrifuged, filtered and
then used to treat H1L1.1c2 cell line, containing a stably
integrated dioxin-response element (DRE)-driven firefly luciferase
reporter plasmid pGudLuc1.1. 24 h after incubation, cells were
lysed and luciferase activity was measured using a luminometer. The
results were normalized based on the negative luciferase activity
of the control.
[0152] Gene Expression Analysis Using Quantitative
Reverse-Transcription PCR.
[0153] RNA isolation, cDNA preparation and qPCR analysis were
conducted as previously described (Lamas et al, 2016). The
oligonucleotides used were as follows: Gapdh (sense)
5'-AACTTTGGCATTGTGGAAGG-3' (SEQ ID No 1) and (antisense)
5'-ACACATTGGGGGTAGGAACA-3' (SEQ ID No 2); Il22 (sense)
5'-CATGCAGGAGGTGGTACCTT-3' (SEQ ID No 3) and (antisense)
5'-CAGACGCAAGCATTTCTCAG-3' (SEQ ID No 4); Reg3g (sense)
5'-TTCCTGTCCTCCATGATCAAAA-3' (SEQ ID No 5) and (antisense)
5'-CATCCACCTCTGTTGGGTTCA-3' (SEQ ID No 6); and Reg3b (sense)
5'-ATGCTGCTCTCCTGCCTGATG-3' (SEQ ID No 7) and (antisense)
5'-CTAATGCGTGCGGAGGGTATATTC-3' (SEQ ID No 8). Gene expression was
analyzed using 2-.DELTA..DELTA.Ct quantification method, with mouse
Gapdh as an endogenous control and the CD group as a
calibrator.
[0154] Glucose Tolerance Test and Insulin Measurement.
[0155] Oral glucose tolerance tests were performed after 11 weeks
of diet. Food and bedding was removed on the onset of the daylight
cycle and mice were treated after a 6 h fasting period with an oral
gavage glucose load (2 g per kg body weight). Blood glucose was
measured before fasting, before oral glucose load and 15, 30, 60,
120 min after oral glucose challenge. Blood insulin was measured
before oral glucose load and 30 min after oral glucose load. Blood
glucose and insulin were determined with a glucose meter (Accu Chek
Aviva, Roche) and Ultrasensitive ELISA kit (Alpco), respectively,
on blood samples collected from the tip of the tail vein.
[0156] Cytokine Quantification.
[0157] Single cell suspensions from spleens were prepared using
mechanical disruption method and then stimulated for 48 h with
phorbol 12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich) and
ionomycin (1 laM; Sigma-ALdrich). Cytokines in the culture
supernatants were quantified using commercial cytokine ELISA kits
(Ebioscience or R&D) or bead-based immunoassay (Biolegend
Legendplex).
[0158] Lamina Propria Cell Isolation and Flow Cytometry.
[0159] Single cell suspensions from the colon and small intestine
lamina propria were isolated and stained as previously described
(Lamas et al, 2016). The following antibodies were used for surface
staining of: CD3 (145-2C11, eBioscience); CD4 (L3T4, BD); CD11b
(M1/70, eBioscience); CD11c (N418, eBioscience); F4/80 (BM8,
eBioscience). Intracellular cytokine staining was performed using
IL-22 (IL-22JOP, eBioscience) and IFN-.gamma. (XMG1.2, eBioscience)
antibodies. The cells were analyzed using a LSR Fortessa cell
analyzer (BD). Lymphocytes were gated using forward scatter (FSC)
and side scatter (SSC).
Example 2
[0160] Here, the inventors show that in both diet- and
genetically-induced animal models of metabolic syndrome, the gut
microbiota exhibits reduced production of AhR ligands.
Supplementation with AhR agonist or Lactobacillus strain with high
natural tryptophan-metabolic activity was sufficient to decrease
the hallmark features of metabolic syndrome, including insulin
resistance and liver steatosis. The mechanisms involved include
correction of the altered intestinal barrier function, a condition
often observed in metabolic syndrome7, and rectification of the
intestinal incretin hormone GLP-1 secretion. Impaired AhR activity
of the microbiota, consistent with lower concentrations of AhR
ligands, was similarly observed in humans with metabolic
syndrome.
[0161] Results
[0162] Products of tryptophan metabolisms are among the key
microbiota-derived metabolites involved in microbiota-host
crosstalk. Indeed, the inventors showed that the inefficiency of
gut microbiota to metabolize tryptophan into AhR ligands is
involved in the pathogenesis of inflammatory bowel disease, notably
through impairment in interleukin (IL)-22 productions. As
intestinal defective IL-22 production was similarly observed in
high fat diet (HFD)-fed mice, the inventors investigated the role
of microbiota-derived AhR ligands in metabolic syndrome. They
observed lower expression of Il22 and other related downstream
genes, such as Reg3g and Reg3b, in the intestine of HFD-fed
compared to control diet (CD)-fed mice (FIG. 1). Colon content of
HFD-fed mice, displaying features of metabolic syndrome including
insulin resistance and hepatic steatosis, showed significantly
lower AhR activity, as assessed by a reporter system, consistent
with reduced concentration of microbiota-derived AhR ligand indole
acetic acid (IAA) and reduced tryptophan concentration in the colon
mucosa compared to CD-fed mice (FIG. 7a-b, FIG. 8). Lower AhR
activity was similarly observed in the colon content of rats fed
with HFD and in murine model of genetically induced metabolic
disorder (leptin-deficient, ob/ob mice) (FIG. 7a). In contrast,
kynurenine, a tryptophan metabolite produced by host cell through
indoleamine 2,3-dioxygenase 1, was significantly increased in HFD
group (FIG. 7c), which is in accordance with the low-grade
intestinal inflammation associated with HFD. Reduced AhR metabolic
activity of HFD microbiota was associated with different profile
compared to CD microbiota, which is reminiscent of previous reports
showing that the microbiota of obese human and animals display
higher Firmicutes and lower Bacteriodetes. Specifically, HFD
microbiota had a relative increase in bacteria belonging to
Lachnospiraceae and Clostridiaceae family while there was a lower
abundance of bacteria from the Rikenellaceae as well as the
Bifidobacteriaceae family compared to CD microbiota.
[0163] To investigate the physiological importance of impaired
microbiota AhR activity, 6-formylindolo(3,2-b)carbazole (Ficz), an
AhR agonist, was administered to HFD-fed mice. Ficz-treatment did
not significantly affect weight gain, but it improved insulin
resistance, as assessed by homeostatic model assessment method
(HOMA-IR), in HFD group (FIG. 9a). HFD-fed mice also showed better
glucose clearance during oral glucose tolerance test (OGGT),
insulin sensitivity during insulin tolerance test (ITT) and
features of non-alcoholic fatty liver disease, including lower
hepatic triglycerides and lower serum concentration of
liver-specific enzyme aspartate transaminase (AST) and cholesterol
(FIG. 9b-c; FIG. 10). Ficz was not able to correct the impaired AhR
agonist production of the microbiota but was sufficient to restore
the intestinal expression of 11-22, Reg3g and Reg3b as well as
Cyp1a1, which is a biomarker for AhR activation (FIG. 11),
highlighting the efficacy of Ficz treatment to compensate for the
reduced microbiota-specific AhR signaling. Moreover, Ficz treatment
was able to reduce glucose dysmetabolism, hepatic dysfunctions and
serum triglycerides in ob/ob mice (FIG. 9e-h, FIG. 12). The
AhR-dependant mechanism of Ficz was confirmed by its lack of
efficacy to treat metabolic syndrome in HFD-fed AhR-/- mice (FIG.
13).
[0164] The inventors next investigated whether administration of a
previously isolated Lactobacilllus reuteri strain that exhibits
natural high tryptophan-metabolic activity can similarly reverse
the HFD-associated metabolic dysfunctions. L. reuteri
supplementation was sufficient to rectify the impaired AhR activity
of HFD-fed mice (FIG. 9i). Furthermore, L. reuteri administration
recapitulated the improvements demonstrated by Ficz treatment,
particularly in regards to glucose clearance, insulin sensitivity
and serum lipid levels (FIG. 9j-m, FIG. 14); thus, underscoring
that microbiota-specific AhR activation is instrumental in
maintenance of metabolic homeostasis.
[0165] Intestinal barrier dysfunction and low-grade inflammation
had been widely accepted as a distinctive feature of metabolic
disorders. Leaky gut allows the passage of microbial products, such
as lipopolysachharide (LPS), across mucosa leading to metabolic
endotoxaemia, defined as moderate increase in plasma concentration
of LPS, a phenotype often observed in humans and animals with
metabolic syndrome and had been further shown to trigger the
systemic inflammatory reaction driving metabolic disease. Indeed,
subcutaneous infusion of LPS induces many features of metabolic
diseases in wild-type mice but not in mice that lacks the LPS
immune co-receptor CD14 and Toll-like receptor 4 knockout mice are
resistant to metabolic dysfunctions. In light of these data, the
inventors sought to determine whether AhR signaling activation has
an impact on intestinal barrier function by evaluating permeability
of different intestinal segments in Ussing chamber. Similar with
previous studies showing barrier dysfunction in HFD-fed animals,
the inventors observed that HFD-fed mice showed higher
translocation of fluorescein-labeled LPS (F-LPS) as well as other
permeability markers, Antonia-red-labeled dextran (ARD4) and
fluorescein-labeled sulfonic acid (FS4), compared to CD group,
particularly in the colon and jejunum. Ficz treatment was effective
in reducing the barrier dysfunction in HFD mice (FIG. 15a-c). To
confirm whether the barrier improvement was specific to Ficz
signaling, they employed a reductionist system involving human
intestinal epithelial cell line. Ficz stimulation was able to
prevent TNF-.alpha.-associated decreased in trans-epithelial
resistance (TER) and increased translocation of fluorescein-labeled
dextran (FD4) in-vitro (FIG. 15d-e). Similar studies demonstrated
the efficacy of Ficz in reversing hypoxia-driven intestinal barrier
dysfunction and the importance of AhR ligands, especially bacterial
derived indole metabolites, in regulating mucosal and epithelial
barrier integrity. Thus, in conjunction with the present results,
these data reinforce the beneficial effect of AhR signaling at
epithelial cell level.
[0166] The inventors next determined the relevance of increased
permeability in HFD-fed mice by measuring serum concentration of
soluble CD14 (sCD14), which is released from monocytes upon LPS
activation and thus can be used as a surrogate marker for systemic
LPS availability. HFD-fed mice showed elevated levels of serum
sCD14 compared to CD-fed mice, and Ficz treatment in HFD-fed mice
significantly prevented this (FIG. 15f). In parallel, Ficz-treated
HFD mice showed reduced systemic inflammatory markers,
characterized by reduced TNF-.alpha. and IFN-.gamma. production by
splenic cells compared to non-treated mice (FIG. 15g-h), suggesting
that reinforcing mucosal barrier may prevent metabolic syndrome
associated endotoxemia and related systemic inflammation.
[0167] In addition to its role in stimulating IL-22 production and
strengthening the integrity of intestinal mucosa, microbiota
derived-indole derivatives had also been shown to be efficient in
stimulating the secretion of the incretin hormone GLP-1 from
intestinal enteroendocrine cells (EEC). GLP-1 has a myriad of
metabolic functions including glucose homeostasis and liver
function, and drugs that mimics GLP-1 action are now widely used in
treatment of type-2 diabetes. The inventors observed significantly
lower expression of intestinal proglucagon, a gene that encodes
GLP-1, and decreased levels of total GLP-1 in the serum of HFD
compared to CD-fed mice (FIG. 15i). Ficz treatment was able to
correct intestinal mRNA expression of proglucagon and serum GLP-1
deficiency in HFD-fed mice, highlighting the physiological
relevance of reduced indole ligands in metabolic syndrome (FIG.
15j). To explore the mechanism leading to correction of GLP-1
following treatment with AhR agonist, GLUTag cells, which is a
murine EEC line that express the proglucagon gene and secretes
GLP-127 as well as highly express AhR, was stimulated with Ficz.
Ficz promoted strong GLP-1 secretion, comparable to when GLUTag
cells were stimulated with Forskolin, a strong inducer of GLP-1
secretion that acts through G protein-coupled receptor. This effect
was confirmed to be AhR specific as the response disappeared in the
presence of AhR antagonist (FIG. 15k). Altogether, the results
suggest a novel mechanism by which AhR signaling may contribute to
the outcome of metabolic dysfunction.
[0168] Finally, the inventors explored whether their findings have
human relevance by analyzing fecal samples from individuals with
metabolic syndrome and from healthy subjects for their ability to
activate AhR (Table 1 for subjects information). Fecal samples from
obese individuals (body mass index, BMI >30) induced lower AhR
activation compared non-obese individuals (FIG. 16a). Furthermore,
AhR activity and BMI showed strong negative correlation (FIG. 16b).
Individuals displaying metabolic risk factors, such as T2D and high
blood pressure (HBP), similarly showed lower AhR activity (FIG.
16c-d, FIG. 17). Fecal samples of individuals with metabolic
dysfunctions further displayed lower concentrations of gut
microbiota-derived AhR agonists, including IAA (FIG. 16e-h, FIG.
18), conforming to the impaired stool AhR activity. In contrast,
fecal kynurenine concentration was up-regulated in individuals with
metabolic dysfunctions (FIG. 16i-l). Collectively, the clinical
results support the relevance of the animal experiments
findings.
TABLE-US-00001 TABLE 1 Clinical subjects information. Patients with
Whole metabolic Healthy population syndrome controls n 127 92 35
Male gender (n, %) 58 (45.7) 43 (46.8) 15 (42.9) Age (mean, SD) 51
(17) 58 (14) 35 (13) BMI (mean, SD) 29 (8) 31 (8) 23 (3) BMI >
30 (n, %) 38 (30) 37 (40) 1 (3) High blood pressure (n, %) 32 (25)
32 (35) 0 Diabetes (n, %) 17 (13) 17 (18) 0
[0169] Materials and Methods
[0170] Mice.
[0171] Male C57BL/6JRj mice and ob/ob mice on the C57BL/6JRj
background were purchased from Janvier (France) and used after 1
week of receipt. AhR.sup.-/- on the C57BL/6JRj background and wild
type mice were housed and bred at Saint Antoine Research Center.
AhR.sup.-/- and C57BL/6JRj at 5 weeks of age were fed ad libitum
with purified control diet (CD, Envigo MD.120508) or high fat diet
(HFD, 18% milk-fat, Envigo MD.97222) for 12 weeks. Ob/ob and
wild-type mice at 6-7 weeks of age were fed ad libitum with
standard chow diet (R03, SAFE, Augy, France) for 12 weeks. 6 weeks
old male Wistar were purchased from Janvier (France), used after 1
week of receipt and fed with either standard chow diet or HFD with
45% of energy from lipids and 17% of energy from sucrose. Animals
were weighed weekly and weekly food consumption was measured
cage-wise. Except when in-vivo permeability experiments were
performed, all animals were fasted for 6 hours prior to sacrifice
and then put to sleep using isoflurane. Animals were culled by
cervical dislocation and appropriate tissues were harvested.
[0172] Animal Treatments.
[0173] For AhR agonist treatment, mice were injected i.p. with
6-formylindolo(3,2-b)carbazole (Ficz, Enzo Life Sciences, 1
.mu.g/mouse) or vehicle (dimethyl sulfoxide, Sigma-Aldrich) once a
week for 12 weeks. For treatment with bacteria with strong AhR
activity, mice were gavaged daily with 10.sup.9 CFU of L. reuteri
CNCM I-5022 or vehicle (MRS broth supplemented with 0.05%
L-cysteine and 15% glycerol) for 12 weeks.
[0174] Measurement of AhR Activity.
[0175] AhR activity of human and animal stool samples was performed
using a luciferase reporter assay method described previously
(Lamas et al, 2016). Briefly, H1L1.1c2 cell line, containing
dioxin-response element-driven firefly luciferase reporter plasmid
pGudLuc1.1, was seeded in 96-well plate and then stimulated with
human or animal stool samples for 24 h. Luciferase activity was
measured using a luminometer and results were normalized based on
the negative luciferase activity of the control.
[0176] Metabolites Measurement.
[0177] Concentration of metabolites from stool samples was
quantified as previously described (Lamas et al, 2016). Briefly,
L-tryptophan and L-kyrunenine were measured via HPLC using
coluometric electrode assay (ESA Coultronics). Indole derivatives
were quantified using liquid chromatography coupled to mass
spectrometry using a Waters ACQUITY ultra performance liquid
chromatography. Concentration of metabolites from stool samples was
quantified as previously described (Lamas et al, 2016). Briefly,
L-tryptophan and L-kyrunenine were measured via HPLC using
coluometric electrode assay (ESA Coultronics). Indole derivatives
were quantified using liquid chromatography coupled to mass
spectrometry using a Waters ACQUITY ultra performance liquid
chromatography (Garner et al, 2007).
[0178] 16s rRNA Gene Sequencing.
[0179] 16s rRNA gene sequencing of fecal DNA samples (collected at
week 9 of CD or HFD) was performed as previously described (Lamas
et al, 2016). Briefly, the V3-V4 region was amplified and
sequencing was done using an Illumina MiSeq platform (GenoScreen,
Lille, Fra). Raw paired-end reads were subjected to the following
process: (1) quality-filtering using the PRINSEQ-lite PERL script
by truncating the bases from the 3' end that did not exhibit a
quality <30 based on the Phred algorithm; (2) paired-end read
assembly using FLASH (fast length adjustment of short reads to
improve genome assemblies) (Schmieder, R. & Edwards, R. Quality
control and preprocessing of metagenomic datasets. Bioinformatics
27, 863-864 (2011)) with a minimum overlap of 30 bases and a 97%
overlap identity; and (3) searching and removing both forward and
reverse primer sequences using CutAdapt, with no mismatches allowed
in the primers sequences. Assembled sequences for which perfect
forward and reverse primers were not found were eliminated.
Sequencing data were analyzed using the quantitative insights into
microbial ecology (QIIME 1.9.1) software package. The sequences
were assigned to OTUs using the UCLUST algorithm (Edgar, R. C.
Search and clustering orders of magnitude faster than BLAST.
Bioinformatics 26, 2460-2461 (2010).) with a 97% threshold of
pairwise identity and classified taxonomically using the Greengenes
reference database (McDonald, D. et al. An improved Greengenes
taxonomy with explicit ranks for ecological and evolutionary
analyses of bacteria and archea. ISME J. 6, 610-618 (2012).).
Rarefaction was performed (13,000 sequences per sample) and used to
compare abundance of OTUs across samples. Alpha-diversity was
estimated using both richness and evenness indexes (Chao1, Shannon
or number of observed species). Beta-diversity was measured by Bray
Curtis distance matrix and was used to build Principal coordinates
analysis (PCoA). Linear discriminant analysis (LDA) effect size
(LEfSe) algorithm was used to identify taxa that are specific to
diet and/or treatment (Segata, N. et al. Metagenomic biomarker
discovery and explanation. Genome Biol. 12, R60 (2011).).
[0180] Oral Glucose Tolerance Test.
[0181] OGGT was performed 5-7 days before the sacrifice. Mice were
fasted by removing the food and bedding 1 hour before the onset of
light cycle. After 6 hours of fasting, glucose solution (2 g/kg for
all mice except; 1 g/kg for ob/ob mice) was administered by oral
gavage. Blood glucose level at time 0 (fasting glucose, taken
before glucose gavage) and at 15, 30, 60 and 120 minutes after
glucose gavage was analyzed using OneTouch glucometer (Roche).
Glucose level was plotted against time and areas under the glucose
curve (AUC) were calculated by following trapezoidal rule. Plasma
insulin concentration (collected in EDTA-coated tubes) at time 0
(fasting insulin) and 30 was analyzed from tail vein blood
(collected in EDTA-coated tubes) using ultra sensitive mouse
insulin ELISA kit (Alpco). Homeostatic model assessment of insulin
resistance (HOMA-IR) was calculated according to the formula:
fasting glucose (nmol/L).times.fasting insulin (microU/L)/22.5.
[0182] Intraperitoneal Insulin Tolerance Test.
[0183] ITT was performed 5-7 days before the sacrifice. Mice were
fasted by removing the food and bedding 1 hour before the onset of
light cycle. After 6 hours of fasting, insulin solution (0.5 U/kg)
was administered intraperitoneally. Blood glucose level at time 0
(fasting glucose, taken before glucose gavage) and at 15, 30, 60
and 120 minutes after insulin challenge was analyzed using OneTouch
glucometer (Roche). Glucose level was plotted against time and
areas under the glucose curve (AUC) were calculated by following
trapezoidal rule.
[0184] Measurements of Plasma Parameters.
[0185] Blood samples were collected in heparin-coated tubes via
cardiac puncture, centrifuged and then plasma samples were stored
at -80.degree. C. until analysis. Plasma cholesterol,
triglycerides, high-density lipoprotein (HDL), aspartate
transaminase (AST) and alanine transaminase (ALT) measurement were
performed by the Biochemistry Platform (CRI, UMR 1149, Paris) using
Olympus AU400 Chemistry Analyzer.
[0186] Liver Histology and Hepatic Triglycerides Measurement.
[0187] A slice of left lobe of the liver was fixed in 4% PFA for 48
h and then transferred to ethanol, fixed in paraffin, trimmed,
processed, sectioned into slices approximately 3 .mu.m thick,
mounted on a glass slide and stained with hematoxylin and eosin.
Hepatic lipids were evaluated and quantified blindly using ImageJ
software as previously described (Schneider et al, 2012; Crane et
al, 2015).
[0188] In-Vivo Intestinal Permeability and Plasma sCD14
Measurement.
[0189] In-vivo assay of intestinal barrier function was performed
using fluorescein-conjugated dextran (FITC-dextran, 4 kDA) method,
as previously described (Laval et al, 2015). Briefly, on the day of
sacrifice, FITC-dextran (0.6 mg/g of body weight) was administered
to the mice by oral gavage and 3 h later, blood samples were
collected in heparin-coated tubes. Fluorescence intensity was
measured in the plasma using a microplate reader (Tecan). Plasma
concentration of soluble CD14 (sCD14) was measured using CD14 ELISA
kit (R&D) as per manufacturer's instructions.
[0190] Intestinal Permeability Measurement in Ussing Chambers.
[0191] Segments of colon, mid-jejunum and distal ileum were cut
along the mesenteric border and mounted in Ussing chambers
(Physiological instruments) exposing 0.2-0.3 cm.sup.2 of tissue
area to 2.5 mL of circulating oxygenated Kreb's Bicarbonate buffer
containing 5 mM KCl, 114 mM NaCl, 2.15 mM CaCl.sub.2, 1.10 mM
MgCl.sub.2, 25 mM Na.sub.2HCO.sub.3, 1.65 mM Na.sub.2HPO.sub.4 and
0.3 mM NaH.sub.2PO.sub.4, and maintained at 37.degree. C.
Additionally, glucose (10 mM) was added to the serosal buffer as a
source of energy and osmotically balanced by mannitol (10 mM) in
the mucosal buffer. Flourescein labeled lipopolysachharide (F-LPS;
80 .mu.g/mL; Sigma-Aldrich) was used as a probe to assess
macromolecular permeability. Additionally, Antonia-red labeled
dextran (ARD4; 400 .mu.g/mL; molecular weight, 4000 Da; TdB) and
fluorescein-labeled sulfonic acid (FS4; 40 .mu.g/mL; molecular
weight, 400 Da; TdB) were simultaneously used to assess
paracellular and transcellular permeability. All probes were added
to the luminal buffer once equilibrium was reached (10-15 minutes
after mounting the tissues in the chamber). Serosal samples (200
uL) were taken at 30 min intervals for 2 h and replaced with fresh
buffer to maintain constant volume. Fluorescence intensity of the
serosal samples was measured using a microplate reader (Tecan) and
concentration of probes was calculated from a standard curve. The
flux of probes from the mucosa to the serosa was calculated as the
average value of two consecutive stable flux periods (60-90 and
90-120 min) and expressed as ng/cm.sup.2/h.
[0192] Monolayer Preparation and TER Measurement.
[0193] Caco-2 cells were grown on Transwell semi permeable filter
support (12 mm diameter wells, polystyrene membranes with 0.4 mm
pores, Costar-Corning), plated at 1.times.10.sup.5 cells per well
and used 18-20 days after confluence. TER was measured at time 0
(TO), which is before adding Ficz (175 nM) onto both the apical and
basal surface 3 h prior to cytokine stimulation and at the end of
cytokine stimulation (time 36 h, T36). Cells were first stimulated
with IFN-.gamma. (10 ng/ml; R&D Systems) for 24 hours to
promote expression of TNF-.alpha. receptors followed by stimulation
with TNF-.alpha. (2.5 ng/ml; R&D Systems) for 12 hours.
Cytokines were only added at the basal compartment without
manipulating the apical compartment. Wells without Ficz and
cytokines were used as controls. TER data was presented as a ratio:
Ratio=(TER Treatment Time 36/TER Treatment TO)/(TER Control T36/TER
Control TO). For flux of fluorescein isothiocyanate-labeled dextran
(FD4; molecular weight, 4000 Da; TdB), monolayers were washed after
stimulation with Hanks' balanced salt solution (HBSS) and
transferred to fresh HBSS. 1 mg/ml of fluorescein
isothiocyanate-dextran was added to the apical layer and incubated
at 37.degree. C. Samples were removed from the basal chamber after
120 minutes. Fluorescence of basal samples was determined using a
fluorescent plate reader (Tecan) and flux was calculated from a
standard curve. Experiments were performed twice in triplicate or
quadruplicate for a total of two independent experiments.
[0194] GLP-1 Secretion.
[0195] GLP-1 secretion was assessed by immunoassay from GLUTag
(Drucker et al, 1994). Cells were plated in 24-well plates at
2.times.10.sup.5 cells per well and cultured for 2-3 days. On the
day of the experiment, cells were washed twice with Kreb's Ringer
solution containing 30 mM KCl, 120 mM NaCl, 0.5 mM CaCl.sub.2, 0.25
mM MgCl.sub.2, and 2.2 mM NaHCO.sub.3 supplemented with 0.5%
(wt/vol) BSA. Cells were stimulated with Forskolin (10 .mu.M;
Sigma-Aldrich) or Ficz (175 nM; Enzo Pharmaceuticals) in the
presence of absence of AhR antagonist CH223191 (10 .mu.M;
Sigma-Aldrich) or vehicle (DMSO) for 2 h in Kreb's Ringer solution.
GLP-1 concentrations at time 0 h and 2 h were measured using a
total GLP-1 Elisa kit (Millipore) as per manufacturer's
instructions. GLP-1 concentrations were expressed as the difference
at time 2 h and 0 h divided by total cell protein concentration.
Experiments were performed twice in triplicate or quadruplicate for
a total of two independent experiments.
[0196] Cytokines Quantification.
[0197] Single cell suspensions from MLN and spleen were isolated by
smashing the cells in 70 .mu.m mesh. 1.times.10.sup.6 cells were
plated in 24 well plate and then stimulated with phorbol
12-myristate 13-acetate (PMA, 50 ng/mL; Sigma-Aldrich) and
ionomycin (1 uM; Sigma Aldrich) for 48 h at 37.degree. C.
Supernatants were collected and used for cytokine analysis.
Cytokines were measured using individual ELISA kit (R&D Mouse
DuoSet IL-6; Mabtech IFN-.gamma., IL-17a ELISA kits; Ebioscience
TNF-.alpha. ELISA kit).
[0198] Gene Expression Analysis Using Quantitative
Reverse-Transcription PCR.
[0199] Total RNA was isolated from different intestinal segments
using RNeasy Mini Kit, according to manufacturer's instructions.
Quantitative RT-PCR was performed using Biorad iScript cDNA
Synthesis kit and then a Takyon SYBR Green PCR kit in a StepOnePlus
apparatus (Applied Biosystems) with specific mouse oligonucleotides
described previously (Lamas et al). qPCR data was analyzed using
the 2.sup.-.DELTA..DELTA.Ct quantification method with mouse Gapdh
as an endogenous control.
[0200] Metabolic Syndrome Cohort.
[0201] All individuals came from three cohorts of Paris Hospitals
(Paris, France) and provided informed consent. All subjects did not
receive antibiotics in the last three months before sampling.
Approval for human studies was obtained from local ethics
committees (Comite de Protection des Personnes Ile-de-France IV,
IRB 00003835 Suivitheque study; registration number 2012/05NICB and
Dispo cohort, registration number 2016/34 NICB; Comite de
Protection des Personnes Ile-de-France III, Mabac cohort,
registration number S.C. 3218).
[0202] Statistical Analysis.
[0203] In each experiment, multiple mice were analyzed as
biological replicates. No statistical methods were used to
predetermine sample size. Dot plots with a linear scale show the
arithmetic mean. Bar graphs are expressed as mean.+-.standard error
of mean (SEM). Except for 16s rRNA results, GraphPad Prism version
7.0b was used for all statistical analysis. The Kolmogorov-Smirnov
test was used to verify that all data set were normally
distributed. For data sets that failed normality, nonparametric
tests were used to analyze statistical differences. For comparisons
between two groups, significance was determined using two-tailed
Student's t-test or nonparametric Mann Whitney test. For
comparisons among more than two groups, one way analysis of
variance (ANOVA) followed by post-hoc Bonferroni test or
nonparametric Kruskal Wallis test followed by post hoc Dunn's test
and two-way ANOVA corrected for multiple comparison with a
Bonferroni test were used. An F or Bartlett's test was performed to
determine difference in variances for t-tests and ANOVAs,
respectively. An unpaired Student's t-test with Welch's correction
was applied when variances were not equal. Differences were noted
as significant at p.ltoreq.0.05.
Example 3
[0204] The inventors assayed the capacity of bacterial strains to
produce AhR agonists (FIG. 19). Bacterial strains CNCM I-5019, CNCM
I-5020, CNCM I-5021, CNCM I-5022, CNCM I-5023 are able to produce
AhR agonists. In addition, other publicly available strains also
show the same capacity.
[0205] Materials and Methods
[0206] Luciferase Assay.
[0207] The H1L1.1c2 cell line, containing a stably integrated
dioxin-response element (DRE)-driven firefly luciferase reporter
plasmid pGudLuc1.1, has been described previously.sup.1,2. The
cells were seeded in 96-well plates at 7.5.times.10.sub.4
cells/well in 100 .mu.l of complete Dulbecco's modified Eagle's
medium (DMEM) (with 10% heat-inactivated FCS, 50 IU/ml penicillin,
and 50 .mu.g/ml streptomycin; Sigma-Aldrich) and cultured
(37.degree. C., 10% CO.sub.2) for 24 h before treatment. This cell
line tested negative for mycoplasma contamination and was used in
this study to determine AHR activity of bacterial samples.
[0208] Bacterial strains were grown in appropriate medium (MRS
medium for Lactobacilli). Culture supernatants were stored at
-80.degree. C. until processing. To assess agonistic activity, the
cells were treated with culture supernatants diluted to 2%, 10%, or
20% in complete DMEM. Controls consisted of cells treated with DMEM
with bacterial culture medium as the negative control, or 0.2
.mu.g/.mu.l of 6-formylindolo[3,2-b]carbazole (FICZ; Sigma-Aldrich)
diluted in DMEM as the positive control. After 24 h of incubation,
wells were washed with 100 .mu.l PBS, and 50 .mu.l Promega lysis
buffer was added to each well. The plates were shaken for 30 min to
lyse the cells. After adding 100 .mu.l of luciferase reagent
(Promega), luciferase activity was measured using a luminometer.
The results were normalized based on the negative luciferase
activity of the control.
REFERENCE
[0209] Crane, et al. Nat Med 21, 166-172, doi:10.1038/nm.3766
(2015). [0210] Drucker, et al. Mol Endocrinol 8, 1646-1655,
doi:10.1210/mend.8.12.7535893 (1994). [0211] Garner, et al. FASEB J
21, 1675-1688, doi:10.1096/fj.06-6927com (2007) [0212] Lamas et al.
Nat Med. 2016 June; 22(6):598-605. doi: 10.1038/nm.4102. Epub 2016
May 9. PubMed PMID: 27158904. [0213] Kerley-Hamilton et al. Environ
Health Perspect. 2012 September; 120(9):1252-9. doi:
10.1289/ehp.1205003. Epub 2012 May 18. PubMed PMID: 22609946;
PubMed Central PMCID: PMC3440132 [0214] Laval, et al. Gut microbes
6, 1-9, doi:10.4161/19490976.2014.990784 (2015). [0215] Schneider,
et al. Nat Methods 9, 671-675 (2012). [0216] Xu et al. Int J Obes
(Lond). 2015 August; 39(8):1300-9. doi: 10.1038/ijo.2015.63. Epub
2015 Apr. 24. PubMed PMID: 25907315; PubMed Central PMCID:
PMC4526411.
Sequence CWU 1
1
8120DNAartificial sequenceprimer 1aactttggca ttgtggaagg
20220DNAartificial sequenceprimer 2acacattggg ggtaggaaca
20320DNAartificial sequenceprimer 3catgcaggag gtggtacctt
20420DNAartificial sequenceprimer 4cagacgcaag catttctcag
20522DNAartificial sequenceprimer 5ttcctgtcct ccatgatcaa aa
22621DNAartificial sequenceprimer 6catccacctc tgttgggttc a
21721DNAartificial sequenceprimer 7atgctgctct cctgcctgat g
21824DNAartificial sequenceprimer 8ctaatgcgtg cggagggtat attc
24
* * * * *